J Clin Pathol: first published as 10.1136/jcp.s1-6.1.22 on 1 January 1975. Downloaded from

J. clin. Path., 28, Suppl. (Ass. Clin. Path.), 6, 22-26

The use of electroimmunoassay for determining specific as a supplement to gel

CARL-BERTIL LAURELL From the Department of Clinical Chemistry, University ofLund, Malmo General Hospital, Sweden

Electrophoretic analysis is the standard method of fraction to the total after scanning or eluting screening for abnormalities of the plasma proteins. the strips and to describe the plasma protein To be acceptable, supportive media should have composition in terms ofthe values found for albumin, negligible adsorption and little interaction with 0x1, OC2-, P-, and y-globulins. Experience of the protein, such as agarose and cellulose acetate. For changes in the electrophoretic fractions in various high quality patterns the plasma proteins should diseases, gained over many years, is an additional separate over more than 5 cm within one hour, requirement for interpretation of the laboratory without the temperature of the supporting medium data. rising above 300. The buffer should be slightly The other method aims at a more profound analy- alkaline, and the addition of calcium (2 mmol/l) sis, and includes, in addition to electrophoresis, the improves the resolution of the three major proteins specific analysis of a limited number of individual in the fl-zone by slowing the f-lipoproteins and the proteins to determine the contribution of thesecopyright. third factor of complement (C3), each to a different proteins to the groups separated by electrophoresis. extent, whereas the mobility of transferrin is unaf- This detects changes in the homeostasis of individual fected. proteins, which are of pathophysiological interest There are two principal methods of interpreting since the metabolic regulation varies from protein to the patterns obtained. One is to relate each major protein. Thirteen major proteins constitute more than 95 % of the total plasma protein (fig 1). An analysis of the contribution made by these proteins to the different electrophoretic zones hashttp://jcp.bmj.com/ been published (Laurell, 1972). Comparison of the electrophoretic pattern with the results of analysis of the major proteins will also indicate whether other minor components, not normally recognizable in the electrophoretic pattern, are increased in a given disease. It is evident that analysis of all 13 proteins will on September 28, 2021 by guest. Protected give more information than measurement of five or six heterogeneous electrophoretic fractions. How- ever, it is by no means certain that the determination of the 13 most abundant proteins is of greater clinical value than that of some 20 other proteins which occur in plasma in such low concentration as not to contribute significantly to the electrophoretic pattern. Everyone who is responsible for the interpretation of electrophoretic patterns must learn to think in terms of specific proteins rather than electrophoretic fractions and to realize how varia- tions in the concentration of different proteins influence the patterns. Fig 1 Electrophoretic distribution of 13 majorplasma It is debatable how many and which of the specific proteins (from Laurell, 1973). proteins should be examined to supplement the 22 J Clin Pathol: first published as 10.1136/jcp.s1-6.1.22 on 1 January 1975. Downloaded from

Electroimmunoassay for determining specific proteins as a supplement to agarose 23 electrophoretic strip in a given patient. The answer if the size of the antigen is small compared with that depends on the clinical problem, the laboratory of the , the latter will substantially influence facilities available, and the cost. the mobility of the complex, and in extreme cases The determination of specific proteins usually may reverse the direction of migration of the antigen. requires immunochemical techniques, namely radial In general this limits the useful size range of antigens immnunodiffusion (RID), electroimmunoassay, or from 40 000 to 4 000 000, but small molecules such automated (AIP). All three are as Ox2-microglobulin have also been successfully sufficiently precise and sensitive for clinical require- measured. The antiserum consumption in the ments. Automated immunoprecipitation is the electroimmunoassay is about 1 ml per 100 analyses quickest and the most expensive, except for large- and the method is more economical for batch scale analysis, and needs the highest quality of anti- analysis because it requires a series of four standards. serum, whereas RID is the slowest, cheapest and Cooled electrophoresis equipment is recommended simplest for the less experienced, although it is since it allows higher voltage (10-15 V per cm) with tedious for examining a large number of specimens. completion of the analysis within three to six hours, Electroimmunoassay occupies an intermediate posi- but less expensive equipment may be used if lower tion in respect of time and costs, and the antiserum voltages and hence longer times are acceptable; a used need not be so very specific; this technique has run with 0.05 M barbital buffer at about 5 V per cm recently been described in detail (Axelsen, Kr0ll, overnight is then usually sufficient. Weeke, 1973), and requires only a brief comment on The decision which of the 13 proteins to deter- the analytical principle. The protein antigen is mine depends on the purpose of the analysis. carried by an electric field into a gel containing Neither scanning of strips nor analysis of specific , the pH and electroendosmosis being proteins are required to recognize myeloma pro- such as to allow little or no migration of the anti- teins, high or low polyclonal IgG, hypoalbumin- bodies to the cathode when the antigen migrates to aemia, signs of inflammatory response, or cirrhotic the anode, or vice versa. Antigen-antibody com- or nephrotic patterns. These and more are recognized

plexes are formed at the front of the migrating anti- at a glance. In fact, for general screening the gel copyright. gen. When the antigen combines with an antibody its pattern is sufficient. However, this gives only a charge decreases and the complex therefore migrates crude assessment, and to assess the degree of more slowly behind the antigen front. abnormality or to study minor changes, quantitative The solubility of the antigen, its molecular size, data are necessary. Acquisition of such data for a and the number of its antibody-combining sites number of proteins is not justified unless the exam- determine the degree of antibody saturation at which iner is experienced in the interpretation of their the complexes will start to precipitate. The optimal significance. Clinicians are unlikely to be able to ratio of antigen to antibody for precipitation in an interpret these data because they are unaware of the http://jcp.bmj.com/ electric field is different from that in free solution. technical difficulties and do not have the advantage Large antigens are precipitated in a zone of large of being able to compare many patterns simul- antigen excess, while smaller antigens migrate until taneously. they approach an antigen-antibody ratio similar to Most clinicians rely on the radiologist to interpret that for precipitation in free solution. However, in their x-ray films, but he requires a clear statement of rocket electrophoresis precipitation always occurs the clinical problem before deciding how to examine with a relatively higher ratio of antigen to antibody the patient and giving his report. Exactly the same on September 28, 2021 by guest. Protected than is required for precipitation of the same system procedure should be adopted for analyses of plasma in solution. A large excess of antigen produces proteins if the maximum of information is to be rockets with a more intense inner contour than obtained. Interpretation of laboratory data in does antigen near the optimal ratio for solutions. pathophysiological terms is usually impracticable The fact that the antigen is only partially saturated in the absence of adequate clinical information. The with antibodies may be utilized for second stage cost of the clinician's time spent in providing such 'sandwich' staining of the precipitates with iso- information on the request form is very much less topically labelled antibodies; this provides a sensi- than the cost of the investigation. tivity down to 1 ,ug per litre as compared with 10 The clinician should state clearly the diagnostic mg per litre for standard electroimmunoassay, the or therapeutic problem. He should also give such latter being sufficient for the determination of most information as length of history, body temperature, plasma proteins. A basic prerequisite for successful and date of trauma or operation etc, otherwise it is application of the method is a difference in charge not possible for the clinical biochemist to decide between antigen and antibody. Antigen size is whether or not, for example, an apparent inflamma- another variable that must be taken into account; tory response (including the immunoglobulin J Clin Pathol: first published as 10.1136/jcp.s1-6.1.22 on 1 January 1975. Downloaded from

24 Carl-Bertil Laurell pattern) fits the case history. The presence of pro- ALBUMIN (BROMCRESOL GREEN) teinuria or gastrointestinal protein loss or the 9, t administration of blood or blood substitutes, of cytotoxic agents, or of steroids should also be stated. The report form should contain the normal ranges of the most commonly analysed of the 13 proteins which constitute more than 95% of the total plasma proteins (fig 1); ca- and ,B-lipoproteins and transferrin need not be included because simple inspection of the gel pattern is sufficient to detect any gross abnormality. The C2-macroglobulin level has, as yet, little clinical significance in spite of its important biological functions. Haemopexin and orosomucoid stain less well than most proteins and are therefore not easily recognized in the patterns. Albumin is routinely analysed separately, usually with bromcresol green (BCG), but examination of 10 20 30 40 50 60 the intensity of staining of the albumin zone will ALBUMIN [IMMUNOCHEMICAL (-ROCKETSf] g/l sometimes reveal probable discrepancies between Fig 2 Comparison between serum albumin values the BCG values and the intensity of the electro- obtained by analysis with bromcresol green and by phoretic band, especially in patients with hypo- electroimmunoassay. albuminaemia. For this reason an immunochemical estimation is needed as a reference method and this, the plasma protein that varies most closely with the because of its sensitivity, is also useful for the analy- erythrocyte sedimentation rate. sis of cerebrospinal fluid and urine. This problem of Haptoglobins and orosomucoid are also reliable

accuracy is evident from fig 2 which compares BCG indicators of an inflammatory response and givecopyright. values and immunochemical albumin values. The few, if any, false positive results. They usually material has been selected from the routine labora- increase in parallel and their synthesis appears to be tory over a year to obtain a large series of hypo- controlled by a common regulator. However, albuminaemic patients. The spread is remarkable in considered individually, haptoglobin is less reliable the low range, where the dye-binding capacity of than orosomucoid because its level may be normal albumin varies markedly in relation to the number or even low as a result of increased catabolism due of antibody-combining sites. Plasma from patients to sepsis, or to decreased hepatosplenic or bone with chronic renal or intestinal protein loss takes up marrow blood flow; furthermore low levels occurhttp://jcp.bmj.com/ a relatively larger amount of the dye per albumin in any disorder with concomitant intravascular content as measured by immunoprecipitation, and haemolysis. Any discrepancy between the levels of this does not develop until after weeks of continuous orosomucoid and haptoglobin may thus be of albumin loss. The chemical background is still diagnostic significance and, in our opinion, both obscure. should be estimated far more often. Their levels As standard indicators of the inflannatory reflect not only the presence of inflammation but response and related reactions we have chosen also its intensity, in acute as well as in chronic on September 28, 2021 by guest. Protected fibrinogen, haptoglobins, orosomucoid, and oxi- conditions. Usually, failure of the haptoglobin level antitrypsin. This may appear overambitious as they to increase at the same rate as orosomucoid indicates usually vary in a similar fashion during infections, a complication. A slight increase of orosomucoid in aseptic necrosis, and after trauma (for references alone may be due to slightly reduced glomerular see Laurell, 1972), but each has its own value in filtration or mild intestinal inflammation. particular cases. Alphal-antitrypsin (oi-AT), which is also an acute Increased fibrinogen is a sensitive indicator of phase reactant, is of special interest in liver disease. inflammation and more sensitive than most others, An absolute or relative increase in oxi-AT occurs, for especially in-chronic diseases affecting the connective examnple, in hepatitis and cirrhosis and contrasts with tissues such as rheumatoid arthritis. It may be the normal orosomucoid and haptoglobin levels in unreliable in acute and subacute inflammation if these conditions. However, such a change is only accompanied by increased fibrinolysis. On the other suggestive of liver disease if the patient is neither hand, slightly increased values are often found pregnant nor taking-oestrogens. In the latter cases without coexisting signs of inflammation in diseases caeruloplasmin also is raised. with increased capillary permeability. Fibrinogen is As warning signs of an inflammatory process, J Clin Pathol: first published as 10.1136/jcp.s1-6.1.22 on 1 January 1975. Downloaded from

Electroimmunoassay for determining specific proteins as a supplement to agarose gel electrophoresis 25

100 200 300 (%) 100 200 300 (%) 01 - 6 AT C~l1-AT

0 100 200 00 (%) 0 100 200 300 (%) OROSO. OROSO. 0 1 1. 2 2.5 */1 w 1- 15 2.5 g/L

HAPTO. HAPTO. 0 1 2 * 5 6 7 910 9/1 2 3 OIL

.~ ' Fl R. 5 6 7 g/L FISR. i sI 91O 5 6 7%i 9 i0 oil 5 A Slight (S) and intense (I) inflam- B Inflammatory response combined C Slight (S) and intense (I) inflamm- matory response, eg, trauma, infection, with increasedhaptoglobin atory response in subacute to chronic or aseptic necrosis. catabolism, eg, septic infection, disease affecting the connective cancer with liver or bone marrow tissues, eg, chronic rheumatoid metastases. The broken line indicates arthritis. the value ofhaptoglobin to be expected in the absence ofincreased catabolism.

0 100 200 300 (%) 200 101 200 100 300 300 %~ 2-AT O1-AT 0~~~~~~0 6 9/1 0l 4 /l

0 100 100 (%) 200 300 (%) 0 300 0 100 200 *300 (%3 ORO SO. OROSO. OROSO. &- A aiII I 0 0.5 0.Q5 t 1.5 2 2.5 9/L 1 1.5 2 2.5 94/ 0 lj 2 2.5 9A copyright.

HAPTO. HAPTO. HAPTO. 0 1 2 3 4 5 6 7 8 0 \ 910 g/t 2 3 6 7 8 910 9/1 1 2.3\42.6 7 Sh0 /I

FIBR. I \ i 4 1 2 FI BS. I| 0 g/L I l I 1 1 > 0 1 6 7 8 9 10 9/1 O 3 6 7 9 10 0 1 2 5 78| 9 10 94

ci CA D response Inflammatory combined E Glomerular protein loss in the F Cirrhosis due to hepatitis B virus, http://jcp.bmj.com/ with fibrinolysis in a heterozygote nephrotic syndrome (broken line = inactive (Ci) and active (CA). for ar-antitrypsin deficiency. The slight loss) broken line indicates values to be expected with inflammation in the absence offibrinolysis in an otherwise normal subject. on September 28, 2021 by guest. Protected O- 100 200 300 *{) C1-AT 4 6 g/l

0 200 300 (%) OROSO. I or 1 I I 0L 1 1.5 2 2.5 g/L Fig 3 Variations ofthe acute phase reactants. The boxes indicate the normal range. HAPTO. 0 1 2 3 4 5 6 7 910 9/1

Fl BR. 0 1 2 5 6 7 8 10 9/1

G Late pregnancy (continuous line) and oestrogen-containing oral contraceptive (broken line). J Clin Pathol: first published as 10.1136/jcp.s1-6.1.22 on 1 January 1975. Downloaded from

26 Carl-Bertil Laurell acute phase reactants fail only on the first day, when not, increased complement activation should be C-reactive protein and antichymotrypsin are already suspected. Measurement ofC3 gives littleinformation increased. unless levels are low, as in diseases with an antigen- With the few exceptions mentioned, orosomucoid antibody reaction in the circulation. C4 is another seems to be the most reliable and the one best complement factor which normally increases during correlated with the extent and intensity of an inflammation. Its variation is of limited clinical inflammatory reaction. If the concentrations of value and it need not be estimated routinely. It often these proteins fall within the normal range, it may disappears selectively in haemolytic anaemias and usually be assumed that there is no inflammatory its measurement is therefore of value when investi- response. This is especially true in patients with gating the cause of reduced haptoglobins. more than one day's fever. However, in chronic Caeruloplasmin is another protein which we have rheumatoid arthritis small nodules and synovitis measured routinely. Its estimation is of very limited may be found without any clear-cut inflammatory diagnostic value but increased levels may suggest the response of the plasma proteins. Varying patterns administration of oestrogens or pregnancy. The of the acute phase reactants are given in figure 3. combination of high oal-AT and caeruloplasmin The importance of the concentrations of IgG, A, helps to distinguish viral hepatitis from liver dys- and M will be discussed by others, but it can be function induced by oestrogens. stated that IgG and IgA should be determined The clinical chemist can indicate not only whether routinely and that their levels should be evaluated the electrophoretic strip and immunochemical in conjunction with the acute phase reactants results are consistent with the clinical diagnosis but taking account of the length of the history. can also interpret the protein changes in terms of The various components of complement must also basic pathophysiological processes. be considered in the discussion of the inflammatory response. We have found that naked-eye inspection References of the electrophoretic strip immediately indicates Laurell, C. B. (1972). Electroimmunoassay. Scand. J. clin. Lab. is normal or not. Since C3 is a slow- Invest., 29, Suppl., 124,21. whether C3 Laurell, C. B. (1973). Electrophoresis, specific protein assays, copyright. or reacting acute phase protein, values may be either both in measurement of plasma proteins? Clin. Chem., 19, 99. normal or increased in acute illness. During subacute Weeke, B. (1973). Rocket immunoelectrophoresis. Scand. J. Immunol., or chronic inflammation C3 should be increased. If 2, Suppl., 1, 37. http://jcp.bmj.com/ on September 28, 2021 by guest. Protected