742 CLINICALCHEMISTRY, Vol. 37, No. 5, 1991

12. Otani G, Abou-el-Makarem MM, Bock KW. UDP-glucurono-syltransferasein perfusedrat liver andin microsomes.Ill. Effectsof galactosamine and carbon tetrachloride on the glucuromda-tion of 1-naphthol and bilirubin. Biochem Pharmacol 1976;25:1293-7.

CLIN. CHEM. 37/5, 742-747 (1991)

13. Peterson GL. A simplification of the protein assay method ofLowry et al., which is more generally applicable. Anal Biochem1977;83:346-56.14.. Shin YS. Diagnosis of glycogenstorage disease. J InheritedMetab Dis 1990;13:419-34.

Immunoturbidimetric Method for Routine Determinations of Apolipoproteins A-I and BDomenico Brustolin,1 Mauro Maierna,’ Francesco Aguzzi,2 Francesco Zoppi,3 Giordano Tarenghi,’ andGiovanni Berti’

A simple immunoturbidimetric method for quantifying apo-lipoproteins (apo) A-i and B in serum or plasma isdescribed. A special reagent formulation, including largeamounts of suitable detergents, obviates the need for asample blank even with grossly lipemic specimens. Theassay is rapid, easily automated, and thus convenient forroutinework. For both apo A-I and apo B, the assay rangeis about 0.2-3.5 g/L. The performance characteristicswere assessed with discrete (Optimate#{174}and Olli CD) andcentrifugal analyzers (Cobas Fara and IL Monarch 2000).Average analytical recovery was 101.5% for apo A-I and99.4% for apo B. Dilution tests showed found/expectedratios of 101.2% (apo A-I) and 101.0% (apo B). Overallprecision (CV) ranged from 1.4% to 3.3% for apo A-I andfrom 1.1% to 8.3% for apo B. Comparisons with commer-cially available rate nephelometry, radial immunodiffu-sion, and immunoturbidimetric methods gave good corre-lations (r �0.938). Using the immunoturbidimetricmethod, we also established the relationships betweenapolipoproteins and lipids and determined the referenceintervals. We conclude that the proposed method issuitable for routine use in clinical laboratories.

AdditionalKeyphrases: “kit” methods - intermethod compari-son

Clinical interest in measuring apolipoproteins hasgrown after the recognition of their physiological impor-tance and utility in assessing an individual’s atheroscle-rotic risk. Some studies suggest that apolipoprotein(ape) A-I and ape B-the major proteins of high-densitylipoproteins (HDL) and low-density lipoproteins (LDL),respectively-are better blood markers for coronary

‘Research & Development Laboratory and Scientific Depart-ment, Bayer Diagnostici SpA, 20040 CavenagoBrianza, Milan,Italy.

2Clinical Chemistry and MicrobiologyLaboratory,Hospital ofBroni and Stradella, 27049 Stradella, Pavia, Italy.

3Clinical Biochemistry and Hematology Laboratory, CaGranda-Niguarda Hospital, 20162 Milan, Italy.

Presented in part at the XIVth International Congress of Clin-ical Chemistry, San Francisco, CA, 1990 (abstract; Clin Chem1990; 36:966)

Received September 4, 1990; accepted March 11, 1991.

heart disease than are the traditional lipid markers(total cholesterol, LDL cholesterol, HDL cholesterol,and triglycerides).4 Some authors today consider the ape

A-I/ape B ratio an even more effective indicator than theindividual apelipeproteins (1-3).

To favor the extension of ape A-I and ape B assaysfrom research or specialized centers to routine clinicallaboratories, we developed an immunoturbidimetricmethod that is simple to use, yet reliable. Based on theformation of immunocomplexes in the presence of Poly-ethylene Glycol 6000, it overcomes many of the techni-cal drawbacks shown by other methods, such as longincubation times and the need for special equipmentand skilled personnel. Like other methods we havedescribed (4, 5), this assay also is particularly suited toroutine work: the test time is short (10-20 mm formanual assays and 5 mm for fully automated assays);no sample blank is needed, even for grossly lipemicsamples; the measurement range is broad (from -0.2 to3.5 g/L for both apelipeproteins); and automation iseasy. Here we describe characteristics and performancesof this method.

Materials and MethodsImmunoturbidimetnc Method

Reagents. The following reagents are available as kits(Sera-Pak#{174}Immuno) from Bayer Diagnostici SpA, Mi-lan, Italy.

1) Antibody reagent: Specific anti-human ape A-I orape B antiserum (from goat) diluted in buffer (Tris, 0.05mol/L, pH 8.0) containing, per liter, 50 g of PolyethyleneGlycol 6000, 1 g of surfactant (Nonfix/95-96; D.A.C.Industrie Chimiche SpA, Milan, Italy), and 1 g ofsodium azide. The reagent is ready for use and stable forat least one year at 2-8 #{176}C.

2) Diluent: Tris buffer (0.01 mol/L, pH 8.0) contain-ing, per liter, 9 g of sodium chloride, 1 g of bovine serumalbumin, 22 g of surfactants (Adekatol SO-105, 10 g,and Adekatol SO-160, 12 g; Asahi Denka Kogyo Co.,Tokyo, Japan), and 1 g of sodium azide.

3) Calibrator: Pooled human serum (with 1 g of

4Nonstandard abbreviations: apo, apolipoprotein; HDL, high-density lipoprotein;and LDL, low-densitylipoprotein.

2.5

2.0

0 5 10

CLINICAL CHEMISTRY, Vol. 37, No. 5, 1991 743

sodium azide preservative per liter), supplied sepa-rately in lyophilized form. Assigned values for ape A-Iand ape B are traceable to the International Union ofImmunological Societies/Centers for Disease ControlApo A-I and Ape B Serum Reference Material (6). Thefive working calibrators (at concentrations rangingfrom -0.2 to 3.5 gIL) are prepared by serial dilution ofthis material with the above diluent and are stable forat least two weeks, both at 2-8 #{176}Cand at 15-25 #{176}C.

Apparatus and assay procedures. For the immunotur-bidimetric assays we used (a) an Optimate#{174}fluorome-ter/photometer (Miles Inc., Diagnostics Division,Elkhart, IN-Gilford Instruments Inc., Oberlin, OH)with a semi-automated procedure: the 21-fold predilu-tion of specimens is carried out separately; (b) an OlliCD discrete analyzer (Kone OY, Espoo, Finland) with asemi-automated procedure; (c) a Cobas-Fara centrifugalanalyzer (F. Hoffmann-La Roche Co., Ltd., Basel, Swit-zerland), with a fully automated procedure; and (d) anIL Monarch 2000 centrifugal analyzer (InstrumentationLaboratory Inc., Lexington, MA) with a fully automatedprocedure.

In all the assays, samples were prediluted 21-fold inthe diluent (with no predilution for the working calibra-tors), the readings were made at 340 nm, and calcula-tion was carried out by the four-parameter logisticmodel or the third-order polynomial function.

No sample blank is required, even for strongly lipemicsamples. However, such samples should be incubated for5-10 mm after the 21-fold dilution and before the assay;this pretreatment makes the sample clear.

In brief, the assay conditions for the semi-automatedprocedures were as follows:

Optimate: mix 10 pL of diluted sample for apo A-I or25 pL for ape B with 500 ML of the respective antibodyreagent; read absorbance after 10 mm at 25 #{176}Cvs thereagent blank (diluent instead of the sample).

Olli CD: mix 8 MLof diluted sample for ape A-I or 20L for ape B with 400 pL of the respective antibodyreagent; read absorbance after 20 mm at 25 #{176}C.

For the fully automated procedures, the assay condi-tions were as follows:

Cobas-Fara analyzer: mix 8 .tL of diluted sample forape A-I or 16 pL for ape B with 12 (ape A-I) or 15 iL(ape B) of water, plus 300 tL of the respective antibodyreagent; the first reading is at 30 s, the second at 5 mm;the reaction temperature is 37 #{176}C.

IL Monarch 2000: mix 10 pL of diluted sample for apeA-I or 20 L for ape B with 50 (ape A-I) or 40 pL (ape B)of water, plus 200 uL of the respective antibody reagent;delay time 5 s; the reaction time is 5 mm at 37 #{176}C.

Comparison Methods

We also assayed ape A-I and ape B by an immunotur-bidimetric method (reagent kits from Eiken ChemicalCo., Ltd., Tokyo, Japan; assays carried out with the ILMonarch 2000 centrifugal analyzer); two rate-nephe-lometry methods (BNA Behring Nephelometer Ana-lyzer and LN reagents, Behringwerke AG, Marburg,Lahn, F.R.G.; and Arraytm Protein System, Beckman

Instruments Inc., Brea, CA); and a radial immunodiffu-sion method (NOR-Partigen#{174}Apolipoproteins A-I and BMonoclonal, plates from Behringwerke AG). The assayswere performed according to the manufacturers’ in-structions.

Blood Lipids and Lipoprotein Cholesterol

We assayed total cholesterol and triglycerides byenzymatic methods (Sera-Pak Cholesterol Fast Colorand Sera-Pak Triglycerides Fast Color kits, both fromBayer Diagnostici SpA). HDL-cholesterol was first sep-arated by the phosphotungstic acid-Mg precipitationtechnique and then assayed enzymatically. To calculateLDL-cholesterol, we used the formula of Friedewald etal. (7).

Results

Optimization of reagents. Because many specimenssubmitted for apolipoprotein assays are lipemic, evenlactescent, we studied a reagent formulation able toremove the intrinsic turbidity without interfering withthe imniunochemical reaction, thus obviating the needfor special chemical pretreatment, long incubationtimes, and measurement of sample blanks. This wasachieved by preparing a diluent of unique composition;it contains, among other things, two noniomc surfac-

tants derived by ethoxylation of linear secondary alco-hols (Adekatol SO Series), at a final concentration of 22g/L. The clarification capacity was demonstrated onlipemic and lactescent sera diluted 21-fold in this di-luent and incubated at 37 #{176}C.Figure 1 shows the clari-fication course. Clear solutions were seen after about 5mm for lipemic sera and after about 10 mm for grosslylipemic sera.

The effectiveness of this diluent was also confirmed intwo other studies. In the first, three pools of clear humansera were enhanced with triglyceride, 10 g/L (Intra-lipid#{174}10%; KabiVitruni AB Stockholm, Sweden), andassayed before (no pre-incubation) and after this addi-tion (10-mm pre-incubation at 37 #{176}C);results of thelatter assay averaged 100.9% of the former assay results

C0

C

0

15

lime, mm

Fig. 1. Clarificationby the diluentUpemic and hyperlipemic humansera (triglycerides from 5.5 to 35 g/L) diluted21 -foldandincubatedat37 C before assay

lime, mm

Fig.2. Kineticsof immunocomplexesformationfor apoA-I(#{149})working calibrators (0.24,0.47, 0.95,1.89,and 3.79 g/L),(A) lipemicsera,and (+) normal sara (fourth andfifthcurves from the t)

0.6 0.4

0.3

0.1

0.0 1.0 20 3.0

ApoA-I, gil.

Fig. 3. Calibrationcurvesfor ape A-I (Ieff) and ape B (right)Readingson Optimate (0) and Cobas-Fara (#{149})analyzers

4.0 0.0 1.0 2.0 3.0 4.0Apo B, gil-

744 CLINICALCHEMISTRY, Vol. 37, No. 5, 1991

for ape A-I and 101.2% for ape B. In the second study,strongly lipemic specimens (triglyceride up to 20 g/L)

were diluted 21-fold and incubated at 37#{176}Cfor 10 miii,and then analyzed along with the respective sampleblanks (prepared by using antibody reagent withoutantiserum). The results were practically the same forthe apolipeprotein concentrations without (y) and with(x) subtraction of the sample blank: for ape A-I, y =

1.OOlx + 0.005 g/L (n = 30, r = 0.999); and for ape B, y= 0.976x + 0.047 g/L (n = 30, r = 0.995). As for theantibody reagent, using a Tris buffer (0.05 molJL, pH8.0) containing Polyethylene Glycol 6000, 50 g/L, en-hanced the formation of the antigen-antibody complex,thus increasing the sensitivity of the reaction. Long-term stability for this reagent (one year refrigerated)was achieved by adding a suitable nomonic detergent, 1g/L, to avoid antiserum precipitation during storage.The antisera were raised in goats injected with purifiedhuman apolipeproteins A-I or B; monospecificity wasassessed by immunoelectrophoresis against pooled hu-man serum. The appropriate antibody dilution, estab-lished for each batch of antiserum, ranged from 1:11 to1:30.

Reaction kinetics. The kinetics of the immunoreactionis of the “quasi-equilibrium” type; the formation ofinsoluble complexes is almost complete after 10 mm.Figure 2 shows the reaction kinetics for working cali-brators and for normal and lipemic sera for ape A-I; the

E0.4

0.2

reaction course for ape B is similar. Although thecomplexes are stable, the reading should be preferablycarried out within 20 min from the start of the immu-noreaction. Temperature in the range 20-37 #{176}Cdoes notremarkably affect the reaction.

Calibration curves. Typical calibration curves ob-tained with Optimate and Cobas-Fara analyzers areshown in Figure 3. The equivalence points were reachedat -10 g/L for both apelipoproteins, a concentration farin excess of clinical findings.

Good repeatability of the calibration curves was foundfor both apelipoproteins on the different analyzers; CVsfor ape A-I and ape B, respectively, were 1.1-5.0% and1.7-4.5% with the Optimate (40 curves over a 20-dayperiod), 4.3-9.6% and 2.9-7.4% with the 0111 CD (eightcurves over a 20-day period), and 2.6-11.3% and 2.2-8.4% with the IL Monarch 2000 (16 curves over a 20-dayperiod).

Linearity and dilution test. To check whether thereagent formulation would affect imniunoreactivity, wecarried out a linearity study with purified (>98%) hu-man ape A-I and ape B (International Enzyme Inc.,Fallbrook, CA), the calibrator of the present method,and two pools of human sera. All these samples werediluted with the diluent to provide decreasing concen-trations of apelipeproteins; we generated the originalcalibration curves with the purified apolipoproteins, andused linear regression to establish correlation and lin-earity. Figure 4 shows good parallelism and linearity ofthe curves, indicating the similar behavior for purifiedand “native” (serum) apolipoproteins. A dilution testwas also carried out by analyzing for both ape A-I andape B three different human sera at various dilutionsand deriving the results from calibration curves gener-ated with the calibrator of the present method (Table 1).

The found/expected ratio averaged 101.2% for ape A-Iand 101.0% for ape B.

Analytical recovery. To three human serum pools weadded various amounts of a serum calibrator at highconcentrations of ape A-I and ape B. Average analyticalrecovery (±SD) was 101.5 (2.0)% (n = 15) for ape A-I inthe range 0.8 to 3.3 g/L, and 99.4 (2.1)% (n = 15) for apeB in the range 0.6 to 2.5 g/L.

Precision. We assessed the present method for preci-

EC0

0.22

1.4

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0Dilution Dilution

Table 1. DIlution Test for Apo A-I and B In Seruma(Found/expected)x 100

Ap0A-I ApoB

Table 2. PrecIsIon Data

Dilution,n-fold A B C D E F

1.2 99.8 99.0 97.0 100.4 97.8 99.41.5 102.1 98.0 100.1 99.8 105.8 100.52 102.5 100.3 100.0 97.8 97.4 100.93 106.4 101.0 101.7 100.9 104.1 101.86 102.1 104.4 101.4 99.4 106.1 103.8

Mean 102.6 100.5 100.6 99.7 102.2 101.3

CV, %

Within-run Between-run Overall

Undlluted values (g/L) forapoA-I:A = 1.173;B = 1.800; C = 1.959; apoB: D = 1.134; E 1.085; F = 1.109.

Ape A-IOptimatea

OlliCDb

IL Monarch2000C

Ape BOptimatea

OlliCDb

IL Monarch2000#{176}

Mean,g/L

0.84 1.55 1.55 2.141.29 2.02 1.16 2.322.01 1.64 1.79 2.441.06 2.08 2.55 3.212.09 2.25 2.44 3.301.34 1.04 0.97 1.492.41 0.91 1.08 1.41

0.48 2.08 1.4.6 2.500.73 2.19 1.64 2.741.22 2.38 1.56 2.050.71 5.63 6.06 8.301.54 4.48 2.86 5.320.62 1.29 3.22 3.541.63 0.92 0.67 1.10

an = 36 each (triplicate assays for 12 runsover a 15-day period); threehuman serum pools.

b = 18 each (triplicate assays for six runs over a 15-day period); twocontrol sara.

= 36 each (triplicate assays for 12 runsover a six-dayperiod);twocontrol sara.

sion, using three analyzers (Optimate, Olli CD, and ILMonarch 2000) to measure ape A-I and ape B in humanserum pools and control sera. Table 2 shows the resultsobtained.

Method comparison. We tested about 200 frozen sera(one freeze-thaw cycle only) covering wide concentra-tion ranges for ape A-I and ape B by the present method,the Eiken immunoturbidimetric kit, and the BehringBNA rate nephelometry method. We also assayed about90 sera (fresh, refrigerated, and frozen) by the presentmethod and by the Beckman APS rate nephelometrymethod. Finally, we tested 50 frozen sera by the presentmethod and by the Bebring radial immunodiffusionmethod. Data were processed by least-squares regres-sion analysis, and the statistical parameters are shownin Table 3. In accord with other authors (8-10), we foundremarkable biases between methods in the ape B deter-mination. As expected, such discrepancies seem mainlyascribable to the different calibration materials used. Infact, when the original data were multiplied by theappropriate factors derived by assaying the calibrator ofthe present method by the comparison methods, theregression parameters for these normalized data mdi-cated (Table 3) that the agreement was quite satisfac-tory.

Interferences. We explored the effect on ape A-I andape B results of adding common anticoagulants inincreasing quantities to pooled normal human sera.

Considering as significant interference a variation±10% ofthe expected apelipeprotein value, we noted nointerference by sodium heparin (up to 120 kilo-mt.units/L), disodium EDTA (up to 8 g/L), sodium fluoride(up to 8,gfL), potassium oxalate (up to 8 g/L), or sodiumcitrate (up to 16 giL). Therefore, plasma can also be usedas sample. Similarly, no interferent effect was found forhemoglobin up to 2 gIL, bilirubin 256 jimol/L, or 98%purified ape A-il (from International Enzyme Inc.), 2g/L. The effect of lipemia has already been discussedunder Optimization of reagents.

Relationship between apolipoproteins and lipid mdi-ces. We tested about 200 human sera for ape A-I and apeB with the present method and also determined totalcholesterol, HDL-cholesterol, and LDL-cholesterol. Wefound significant correlations (P <0.00 1) between ape

2.1

CLINICALCHEMISTRY, Vol. 37, No. 5, 1991 745

0.7

Fig. 4. Linearity(!ef Apo A-I: (#{149})= pureapo A-I; (A) = calibrator of the present method, y = 2.030x + 0.023g/L (r = 0.999, S = 0.013); (0) = liquid human serum pool, y= 2.204x + 0.020 g/L (r = 0.999, Sr,. = 0.009). (nghl) Ape B: (C) = pure ape B; (A) = calibratorofthe present method,y = 2.632x + 0.010 g/L (r 0.999, S= 0.007); (0) = liquid human serum pool, y = 2.625x + 0.014 g/L (r= 0.999, S = 0.014)

Table 3. CorrelatIon Data

746 CLINICALCHEMISTRY, Vol.37, No. 5, 1991

Present method (j4

andInstrument

Ape A-IIL Monarch2000

IL Monarch2000

OlliCD

Optimate

Apo BIL Monarch2000

IL Monarch2000

OIIi CD

Optimate

Comparison method andInstrument

Ratenephelometry,BehnngBNA

Eikenimmunoturbidimetry,IL Monarch2000

Rate nephelometry,BeckmanAPS

Behringradialimmunodiffusion

Rate nephelometry,BehnngBNA

Eiken immunoturbidimetry,IL Monarch2000

Ratenephelometry,BeckmanAPS

Behringradialimmunodiffusion

n r Slope

197 0.961 1.068

198 0.928 0.984

85 0.966 0.892

50 0.967 0.811

54)6 0.967 0.956

197 0.941 0.6641976 0.941 1.117199 0.938 0.5971996 0.938 0.835

85 0.955 0.672856 0.944 0.76650 0.948 0.57650 0.948 0.910

Intercept S,,, 3;

g/L

Range forx

-0.087 0.091 1.56 1.58 0.65-2.46

0.047 0.124 1.56 1.58 0.70-2.54

0.157 0.087 1.31 1.32 0.54-2.42

0.072 0.092 1.44 1.24 0.55-2.15

0.073 0.093 1.23 1.25 0.48-1.85

-0.021 0.088 1.68 1.09 0.81-3.18-0.021 0.088 1.00 1.09 0.48-1.89

0.153 0.090 1.57 1.09 0.62-3.180.153 0.090 1.12 1.09 0.44-2.27

0.222 0.083 1.05 0.92 0.30-2.070.231 0.091 0.91 0.93 0.26-1.80

0.058 0.066 1.06 0.67 0.48-2.300.058 0.066 0.68 0.67 0.30-1.42

Bias,g/L (and %)

0.02 (1.3)

0.02 (1.3)

0.01 (0.8)

-0.20 (-13.8)

0.02 (1.6)

-0.59 (-35.1)0.09 (9.0)

-0.48 (-30.6)-0.03 (-2.7)

-0.13 (-12.4)-0.02 (-2.2)

-0.40 (-37.7)-0.01 (-1.4)

6rTnallzod data.

A-I (y) and HDL-cholesterol (x): y = 0.735x + 0.60 g/L (r= 0.844; n = 199; = 1.35 mmol/L,5; = 1.59 g/L;x range= 0.57-2.38 mmol/L); ape B (y) and LDL-cholesterol (x):y = 0.168x + 0.26(r = 0.877; n = 191; = 4.92,5; = 1.09;x-range = 1.42-9.79); and ape A-I/ape B (y) and HDL-cholesterollLDL-cholesterol (x): y = 3.767x + 0.4 (r =

0.875; n = 190; = 0.30,5; = 1.54; x-range = 0.08-0.86).There was no significant correlation between ape A-I (y)and total cholesterol (x): y = 0.021x + 1.44 (r = 0.081; n= 196; = 7.07, .3;= 1.59; x-range = 3.70-11.99),whereas the correlation between ape B (y) and totalcholesterol (x) was significant: y = 0.168x - 0.09 (r =

0.875; P <0.001; n = 194; = 7.07,5; = 1.09). These dataagree with those previously reported (8, 10).

Storage of samples. As recommended for each pro-posed method (9), we evaluated the valid period ofstorage for ape A-I and ape B for assay by the presentmethod. After the initial assay, aliquots of about 30different sera were stored refrigerated at 5#{176}Cfor oneweek and at room temperature (20-25#{176}C)for 12 h;moreover, the samples prediluted 21-fold were stored for6 h at room temperature and then re-assayed. Closeagreement between apelipeprotein concentrations be-fore (y) and after (x) storage was found for both ape A-Iand ape B. For example, the regression parameters forape A-I, for refrigerated samples, were y = 1.046x -

0.022 g/L (n = 30, r = 0.997); for samples at roomtemperature,y = 0.988x + 0.017 g/L (n = 20, r = 0.994);and for diluted samples, y = 0.949x + 0.02 1 g/L (n = 20,r = 0.998).

Long-term stability studies were also carried out ontwo pools of human sera stored at -20 #{176}C;no significantchange in ape A-I and ape B concentrations was foundafter at least three months. The repeated freezing and

thawing process randomly caused notable variations inapelipeprotein concentration; therefore, samples frozenmust be thawed only once before analysis.

Reference intervals. The reference intervals for thetwo apolipoproteins were calculated after assaying bythe present method 135 normolipemic EDTA-plasmasamples (total cholesterol <6.21 mmol/L and LDL-cho-lesterol <4.14 mmol/L). Gaussian distribution was as-certained (11) for both analytes. The 95% parametric

intervals were 0.89-1.98 (mean 1.43) g/L for ape A-I and0.30-0.95 (mean 0.62) g/L for ape B.

DiscussIon

Increased clinical interest in assessing concentra-tions of plasma apolipoproteins calls for methods thatwill be suitable for most clinical laboratories. To ftillyexploit their diagnostic potential, assays of apolipopro-teins should also be convenient for a wide applicationin the routine laboratory. Many immunological tech-niques have been used for quantifying apolipoproteins.Radioimmunoassay and enzyme-linked immunosor-bent assay are specific, but too sensitive, time-consum-ing, and not automated. Electroimmunoassay is con-fined to specialized centers; radial immunodiffusion is a

simple method particularly suited to small laboratoriesbut requires long incubation times. Immunonephelom-etry and immunoturbidimetry are greatly appreciatedin routine work because they are more suited to auto-mation; moreover, whereas the former technique stillappears to be affected by lipemia, the latter is reliablewith any type of sample. Immunoturbidimetry is alsoattractive because it requires neither dedicated instru-mentation nor skilled personnel; rather, it is easily

CLINICALCHEMISTRY, Vol. 37, No. 5, 1991 747

adapted to plain spectrophotometers and automatedanalyzers.

We developed an immunoturbidimetric method thatproved to be simple, rapid, and easy to use for measur-ing ape A-I and ape B. Being easily adaptable to manyinstruments, it is suitable for analyzing small andlarge batches of specimens. Many reports (12, 13) havedealt with the need to pretreat samples to maximizethe exposure of antigenic sites (ape A-I) or to minimizeartifacts in the presence of high concentrations oftriglycerides (ape B). The particular formulation of ourreagents allows a complete immunoreactivity with nosignificant influence by hypertriglyceridemia. Samplepretreatment is therefore usually not required; forgrossly lipemic specimens, a short pre-incubation (5-10mm at 37 #{176}C)with a diluent containing detergents thatallow maximum exposure of epitopes without any neg-ative influence on their reactivity is enough for clari-fication. The method is precise and free of interfer-ences.

The correlation studies showed for ape A-I closeagreement and negligible biases with commercially

available nephelometry and turbidimetry methods;with the radial immunodiffusion method, a moderatebias was observed. For ape B, correlation coefficientswere still excellent but biases were always important.Discrepancies among methods are not unusual in ape-lipeprotemn assays and may be method-dependent orrelated to different reactivities of calibration materials.Serum pools in lyophilized form are valid as calibratorsfor ape A-I measurements but are not suitable for ape Bmeasurements because of matrix effects among methods(14, 15). We found that our biases between methodswere mostly due to the different calibrators used by thevarious manufacturers, confirming once again the needfor a common reference material, especially for ape Bassays (9).

Substantial reduction in intermethod and interlab-oratory variations-essential before researchers can de-fine common reference intervals and cutoff points forclinical use-will be achieved only when a common andinternationally recognized reference material is madeavailable. This is expected when the ongoing Interna-tional Federation of Clinical Chemistry apelipoproteinstandardization program is completed.

In conclusion, the method at issue, besides beingreliable, is convenient and suitable for use in all routine

clinical laboratories, thus fulfilling the typical require-ments for measuring biochemical markers in the gen-eral population.

The valuable contribution of Dr. A. Fumagalli (Bayer Diagnos-tici SpA) in statistical analysis has been appreciated.

References1. Kottke BA, ZinsmeisterAR, Holmes DR,et al. Apolipoproteinsand coronary artery disease. Mayo Clin Proc 1986;61:313-20.2. Naito IlK. The clinical significance of apolipoprotein measure-ments. J Clin Immunoassay 1986;9:11-20.3. Bachorik PS, Kwiterovich P0 Jr. Apolipoprotein measure-ments in clinical biochemistry and their utility via-a-vis conven-tional assays. Clin Chim Acta 1988;178:1-34.4. DonA V. Papagni M, Tarenghi G, Aguzzi F, Berti G. Specificserum protein quantitation by a simple iznmunoturbidimetricmethod. Giorn It Chim Clin 1987;12:205-14.5. Croci D, Nespolo A, Bosom MA, Tarenghi G. A simple immu-noturbidimetric method for IgG and albumin quantitation incerebrospinal fluid and serum. J Clin Chem Clin Biochem 1989;27:863-S.6. Smith SI, Cooper GR, Henderson LO, Hannon WH, the Apo-lipoprotein Standardization Collaborating Group. An interna-tional collaborative study on standardization of apolipoproteinsA-I and B. Part I. Evaluation of a lyophilized candidate referenceand calibration material. Clin Chem 1987;33:2240-9.7. Friedewald WT, Levy RI, Fredrickson DS. Estimation of theconcentration of low-density lipoprotein cholesterol in plasma,without use of the preparative ultracentrifuge. Clin Chem 1972;18:499-502.8. MaciejkoJJ, Levinson SS,Markyvech L, Smith MF, Blevins It.New assay of apolipoproterns A-I and B by rate nephelometryevaluated. Clin Chem 1987;33:2065-9.9. Marcovina SM, Albers JJ. Standardization of the immuno-chemicaldetermination of apolipoproteinsA-I and B: a report ofthe International Federation of Clinical Chemistry Meeting onstandardization of apolipoproteinA-I and B measurements (Basisfor future consensus), Vienna, Austria, April 18-19, 1989. ClinChem 1989;35:2009-15.10. Mount JN, Kearney EM, RosseneuM, Slavin BM. Immuno-turbidimetric assaysfor serum apolipoproteins A-I and B usingCobasBio centrifugal analyzer. J Clin Pathol 1988;41:471-4.11. Solberg HE. The theory of referencevalues. Part 5. Statisticaltreatment ofcollected reference values. Determination ofreferencelimits. J Clin Chem Clin Biochem1983;21:749-60.12. Steinberg KK, Cooper GR, Grainer SR. Rosaeneu M. Someconsiderations of methodology and standardization of apolipopro-tein A-I imxnunoasaays. Clin Chem 1983;29:415-26.13. Rosaeneu M, Vercaemst R, Steinberg KK, Cooper GR. Someconsiderationsof methodologyand standardization of apolipopro-thin B immunoassays. Clin Chem1983;29:427-33.14. Smith SI, HendersonLO, Harmon WI!, Cooper GR. Effectsofanalytical method and lyophilized sara on measurementsof apo-lipoproteins A-I and B: an international survey. Clin Chem1990;36:290-6.15. Marcovina SM, Adolphson JL, Parlavecchia M, Albers JJ.Effectsof lyophilization of serum on the measurement of apolipo-proteins A-I and B. Clin Chem 1990;36:366-9.