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FTC-insulin conjugates. I. Preparation of fluorescein thiocarbamyl-insulin conjugates
Journal of Immunological Methods 2 (1973) 371- 382. © North-Holland Publishing Company
FTC--INSULIN
CONJUGATES.
I. P R E P A R A T I O N OF F L U O R E S C E I N
THIOCARBAMYL-INSULIN CONJUGATES
A. JOBB/kGY and G.M. JOBBAGY Laboratory o f lmmunopathology, Hungarian State Institute o f Dermatology and Venereology, Budapest, Hungary
Received 15 February 1972
Accepted 30 June 1972
Fluorescein thiocarbamyl-insutin conjugates were prepared, partly for the immunofluorescent technique, partly to study the labelling process. The quality of the raw materials and the characteristics of the FITC-insulin reaction were investigated. Labelling was carried out with a calculated amount of FITC. This technique enabled us to control the labelling process and to obtain conjugates with most favourable characteristics.
1. INTRODUCTION Fluorescein t h i o c a r b a m y l - i n s u l i n conjugates are used to demonstrate intracellular insulin antibodies in patients with diabetes mellitus (Berns and Blumenthal, 1962; Berns et al., 1962; Titze et al., 1962; Parker et al., 1963). Since antibodies develop after treatment with bovine or swine insulin, a mixture containing both insulin preparations was labelled with fluorescein isothiocyanate (FITC) for use in the tests. Labelling was carried out with crystalline FITC isomer I. The characteristics of the FITC preparation were studied in order to obtain information about the dye and isothiocyanate content. These data made it possible to ensure the most favourable degree o f labelling. Theoretically i mol o f FITC is required to label 1 mol of insulin. In practice, however, a little more dye is needed because of the incomplete homogeneity of the labelling process. Ratios from 1 - 1 . 5 may be regarded as most favourable, with less influence on the biological activity o f insulin than higher values. In this paper a m e t h o d is suggested for the preparation o f F T C - i n s u l i n conjugates, with o p t i m u m labelling efficiency.
372
A. JOBB,~GY and G.M. JOBBA,GY
2. MATERIALS AND METHODS
2.1. lnsulin A mixture of porcine (mol.wt. 5777) and bovine (mol.wt. 5733) preparations, was obtained from Kt3bhnyai Gydgyszer~rugy~r (Budapest); for the calculations an average mol.wt, of 5755 was employed. The preparation had been recrystallized 10 times and its activity was 23 units/rag.
2.2. Reagents Crystalline fluorescein isothiocyanate isomer I (mol.wt. 389.39) was purchased from SPOFA (Czechoslovakia). 0.5 M carbonate buffer pH 9.0, containing Na2 CO3 (anhydrous) 1.2 g; NaHCO3 7.4 g; distilled water to 200 ml; a 1 : 10 dilution was used for gel filtration.
2.3. Determination of the dye content of conjugates Dye content was determined by direct measurement of optical density at 496 nm in 0.1 N NaOH (Jobb~gy and Jobb~igy, 1973a, b).
2.4. Protein determittation of conjugates The protein determination was carried out according to the Biuret method. Extinction was measured at 565 nm and the value obtained corrected for absorption by the dye at tile same wavelength.
3. EXPERIMENTS Three samples of insulin were labelled in parallel with FITC. The volume of each conjugate was 5 ml and the protein content 15 mg/ml; their expected respective molecular ratios FTC : insulin were: 1.05, 1.22 and 1.40 : 1. To ensure the desired degree of labelling the following preliminary experiments were carried out.
3.1. Determination of the dye content of the fluoresce& isothiocyanate preparation FITC preparations often contain a certain amount of colourless impurities and/ or preserving agents. It is therefore necessary to determine the total dye content. 5 mg dye was dissolved in 100 ml 0.1 N NaOH. The solution was diluted 1 : 25 and readings were made at 488 nm and 320 nm. A solution containing 1 /ag/ml fluorescein was used as reference standard. Dye content was calculated according to the following formula:
FTC-insulin conjugates. I
cd -
Q'F1TC QFITC
373
FMwl "FMel ' CF" Vd' Dd" EFITC 488 corr. QFITC " EF 489 corr.
•
103
2.40735 • EFITC 488 corr.
(1)
where -- The dye content of the FITC preparation;
ca
= The quantity of the dye determined photometrically, in mg; = The total amount of dye employed for the dye content determination, in mg (5 mg); FMwL = The ratio of the molecular weights of F1TC and F (1.1721); FMe 1 = The ratio of the molecular extinction coefficients of F and FITC (0.961218); CF -- The concentration of the fluorescein standard solution, in/ag/ml (1/ag/ml); -- The volume of the dye solution (100 ml); Dd = The dilution of the dye solution employed for the photometric readings (1 : 25); EFITC 488 corr. = The corrected extinction of the FITC solution at 488 nm wavelength (0.2077); The corrected extinction of fluorescein at 489 nm wavelength EF 489 corr. = (0.234). The dye content of SPOFA FITC isomer I was found to be 0.984. O'FITC QFITC
3. 2. Determination o f the optimum labelling efficiency The dye content gives information about the ratio of the coloured constituents of dye products. This value, however, is not equal to the FITC content, because the side- and degradation products of FITC are also coloured. The true FITC content varies according to the type of product and the duration and condition of storage. The determination of the optimum labelling efficiency is an arbitrary measure which provides an indication of the true FITC content of the dye products. 5 ml albumin (50 mg/ml) was labelled with 1 mg SPOFA FITC at 25°C for 24 hr. The albumin conjugate was gel filtered and the FTC content determined. The ratio bound FTC : added dye (coloured ingredients) expresses the optimum labelling efficiency. CFTC corr. a ' D1 a " Va Col
QFITC " Cd" 103
374
A. JOBB,~GY and G.M. JOBB,~GY FMwl" FMw2" FMe2a ' CF ' D l a ' D2a" Cpa" Va" EFTC 492 corr. Dla" QFITC" Cd" %ca" 103"E F489 con.
(2)
= 2.62577 - EFTC 492 corr. whe re:
= The optimum labelling efficiency; = The dye content of the FTC albumin conjugate, corrected for loss of dye during gel filtration, in/ag/ml; The amount of FITC added (1.0165 rag); QF1TC = The ratio of the molecular weights of FTC radical and FITC FMw2 = (1.0026); The ratio of the molecular extinction coefficients of FITC and FMe2a FTC radical bound to albumin (1.856); The dilution of the FTC-albumin conjugate for photometric O2a = readings applied to the dye content determination (1 : 25); a = The protein content of the albumin solution (50 mg/ml); Va = Volume of the albumin solution (5 ml); The protein content of the FTC-albumin conjugate (21.27 mg]ml); ca EFTC 492 corr. = The corrected extinction of the F T C - albumin conjugate at 492 nm wavelength (0.3455). The optimum labelling efficiency, using SPOFA FITC, was 0.922.
Eo I CFTC corr. a
3. 3. Determination of the efficiency of labelling Three parallel samples of insulin (5 ml-15 mg/ml) were labelled with different amounts of FITC (table 1) at 25°C for 4 hr. Under these circumstances the reaction is usually not complete. The ratio bound FTC : FITC added expresses the efficiency of labelling.
Table 1 Determination of the labelling efficiency of FTC insulin conjugates. Conjugate
FMe2i
EFTC 490 corr. QFITCmg
Cpic mg/ml
El
1
1.1002
0.2725
8.5294
4.1606
0.6836
2
1.1339
0.3410
9.9509
4.3173
0.7283
3
1.0539
0.3970
11.3725
4.2851
0.6948
Average
0.7022
FTC-insulin conjugates. I
375
CFTC corr. i " Vi ' D 3
E1 QF1TC" Cd "Eo I " 103 FMwl" FMw2 " FMel " FMe2i " DI i " D2i " Cpi • Vi " EFTC 490 corr. D1 i" QFITC " Cd " Eo 1 " Cpic" 103 • E F 489 corr.
= 80•9207
FMe2i "EFTC 490 corr.
(3)
QFITC " Cpic where: = Labelling efficiency of the insulin; = The dye content of the FTC-insulin conjugate, corrected for loss of dye during gel fltration; vi = Volume of the insulin solution used for labelling (5 ml); FMe2i = The ratio of the molecular extinctions of FITC and FTC bound to insulin (table 1); D2i = The dilution of the FTC-insulin conjugate used for photometric readings (1 : 200); Cpi = The protein content of the insulin solution used for the labelling, in mg/ml (15 mg/ml); Cpic = The protein content of the insulin conjugate, in mg/ml (table 1); EFTC 490 corr. = The corrected extinction of the FTC-insulin conjugate at 490 nm wavelength (table 1). The labelling efficiencies for insulin were similar in the three conjugates and showed an average o f 70%. In the range 1 - 1 . 5 mol of dye to 1 tool of protein, the efficiency of labelling was not affected by the d y e - p r o t e i n ratio.
El CFTC corr. i
3•4. Calculation o f the desired degree o f labelling Insulin has 3 free amino groups which are able to react with FITC in an additional reaction. Theoretically at least 1 FITC mol is needed for 1 mol of insulin• A higher dye ratio is not recommended, because of its effect on the biological activity of insulin. The labelling is not homogeneous - some insulin molecules bind two FTC radicals and some none at all. To ensure the labelling of all insulin molecules, the dye ratio was increased above 1. The FITC ratios of the three parallel samples were: 1.05, 1.225, 1.40. For the calculation o f the amount of FITC necessary, the desired degree of labelling must be expressed as a weight ratio.
376
A. JOBB,~GY and G.M. JOBB,~GY CFITC
Dtw 1
(4)
Cpi
where: Dlw I = The desired degree of labelling, in terms of a weight ratio; CFITC = The amount of FITC added, in/ag/ml; Cpi = The insulin content of the solution, in mg. So far only the molecular ratio of the desired degree of labelling has been determined; the weight ratio can be calculated from this value by the following formula: CFITC Dlml
=
MWFITC. 10 3-
=
Cpi
CFITC • Mw. l Cpi" MWFITC • 103
(5)
Mw i where: Dim I
= The desired degree of labelling, in terms of a molecular ratio; = The molecular weight of insulin. CFITC in equation (5) is substituted in equation (4).
Mw i
Dim 1 ' Cpi" MWFITC - 10 3 CFITC
Dim 1 • Cpi" MWFITC • 10 3 Dlwl
(6)
Mw i
Cpi" M w i
Dim 1 • M W F I T C
•
103
Mw i
= 67.6612 • Dlm l
(7)
where: MWFITC
= The molecular weight of FITC (389.39); = The molecular weight of insulin (5755). The results are listed in table 2.
Mw i
3. 5. Calculation o f the amount o f F I T C used f o r the labelling o f insulin
Three 5 ml samples of an insulin solution (15 mg/ml) were labelled with FITC. From the above data the necessary amount of dye was calculated according to the following formula:
FTC-insulin conjugates. I
377
_ Cpi" Vi" Dlw1
QFITCi
-
E1 . Eol" Cd" 103
(s)
= 0.12005 - Dtwl
where: QHTCi = The amount of FITC used for the labelling of insulin, in mg. For other symbols see the previous equations. The results are listed in table 2. Table 2 The desired degree of labelling. Conjugate
The desired degree of labelling expressed as: molar ratio Dlml
weight ratio Dlwl
The amount of FITC used for labelling QFITC
1
1.050
71.0442
8.5288
2
1.225
82.8849
9.9503
3
1.400
94.7256
11.3718
3. 6. Labelling o f insulin with FITC 375 mg insulin were dissolved in 10 ml 0.03 N HCL, 10 ml physiological saline and 5 ml 0.5 M Na2 CO3 buffer (pH 9.0) in this order. The final concentration of insulin was 15 mg/ml. 5 ml of this stock solution were placed in a 25 ml Erlenmeyer flask. For each sample a 3 ml FITC solution was prepared with the necessary amount of FITC per ml (table 2). 1 ml was measured with an Autospencer for the labelling o f the insulin, and 1 ml was used for the FMe2i factor determination. The dye solution was added to the insulin solution at room temperature drop by drop, with continuous stirring. The flasks were then put in a Vibrotherm and shaken for 4 l~r at 25°C. Unbound dye was removed on a Sephadex G-25 (fine) column equilibrated with 0.05 M carbonate buffer. The volume of the column was 4 times the volume of the insulin solution. During gel f'dtration part o f the conjugate remained in the gel. This loss of insulin can be calculated from the original protein content (Cpi) and the protein content of the conjugate (Cpic), according to equations 9 and 10. % i loss
= C p i - %ic "D3
% i loss %
_ Cpiloss" 100 Cp i
(9)
(10)
Conjugates
Protein content of conjugates Cpic
4.1606 4.3173 4.2851
Protein content of insulin solution of mg/ml
15
15
15
2.8382
2.9218
3.0887
Dilution of conjugate D3
12.1620
12.6143
12.8508
Protein content related to the original volume Cpic.r.
Table 3 Determination of the protein loss of insulin conjugates.
2.8380
2.3857
2.1492
mg/ml
18.920
15.905
14.328
%
Loss of insulin
1.4
1.225
1.05
Degree of labelling
,<
~a e~
~,, C~
oo
FTC-insulin conjugates. I
379
where: Cpi loss
= The loss of insulin, in mg/ml;
Cpi loss %
= The loss of insulin, in percent.
The protein loss varied from 14-19%, and increased proportionally with the degree of labelling (table 3). The conjugates were characterized chemically and freezedried. The methods used for chemical characterization will be published in a subsequent paper (Jobb~gy and Jobb~gy, 1973c).
4. RESULTS AND DISCUSSION The aim of our experiments was to prepare a reagent for the demonstration of intracellular anti-insulin antibody in patients treated with insulin of various animal origins and to study the labelling reaction. Pure crystalline insulin, being chemically homogeneous, is an ideal substance for investigating the labelling process. In order to obtain a high quality reagent, the following aspects of the process have been studied: 1. The quality of the dye preparation; 2. The characteristics of the protein solution to be labelled; 2. The characteristics of the FITC-protein reaction; 4. The desired degree of labelling.
4.1. The quality o f the dye preparation Manufacturers of dye preparations do not all supply data about the quality of their products. In the majority of cases these are contaminated with coloured and colourless impurities to a greater or lesser extent, depending upon the grade of preparation and the duration and condition of storage. A certain amount of information can be obtained from the following: a. Determination of the dye ratio (coloured : colourless ingredients); b. Determination of the ratio FITC : dye; c. Calculation of the actual FITC content of the preparation. The methods for the determination and the data for SPOFA FITC isomer I are outlined in sections 2 and 3. Several dye preparations were compared. Results and details of the methods have been published (Jobb~gy and Jobb~gy, 1973a,b). Both methods supply useful information on the quality and the purity of dyes. The true FITC content may be calculated by multiplying the two ratios. Even crystalline FITC preparations need chemical characterization, because during their storage the FITC content decreases. Thus, the preparation we employed contained only 90% FITC.
380
A. JOBB~,GY and G.M. JOBB,~GY
4.2. The characteristics o f the prote& solut&n selected for labelling Volume, protein content and biological activity must be determined before labelling. The inorganic contamination of the crystalline insulin preparation was very low, therefore the protein content was not examined.
4. 3. The characteristics o f the FITC protein reaction 4.3.1. Speed of reaction Several factors may have an effect on the reaction speed:
a. Concentration o f insulin An insulin concentration of 15 mg/ml was selected for the labelling, because this value lies within the normal protein concentration range.
b. Concentration o f FITC The concentration of FITC employed was determined by the protein concentration and the desired degree of labelling. The latter was somewhat more than 1 tool dye/1 mol insulin, but the values were close to each other. Therefore, the dye concentration did not play a significant role in the modification of the reaction speed over the range employed.
c. Temperature o f the reaction m&ture 25°C was selected for labelling, although 4°C has been suggested by several authors. At that temperature, however, the reaction speed is very low. For practical and economical reasons - shorter labelling duration, smaller amount of dye - the warm labelling process was chosen.
d. Agitation o f reaction m&ture The Vibrotherm provided a constant temperature and continuous shaking of the reaction mixture. Thus the reaction speed was increased, and one could expect a more homogeneous distribution of the FITC molecules.
e. pH o f the reaction mixture Generally pH 9.0 is used. At a lower pH the reaction speed is very slow, while at higher pH the biological activity of insulin may be decreased.
FTC-insulin conjugates. I
381
4.3.2. Duration of the labelling process 25°C is a favourable temperature for some bacterial growth, therefore a reaction time of 4 hr was employed. 4.3.3. Efficiency of labelling During 4 hr of labelling the reaction is not complete. Under these conditions only a proportion of the dye molecules are able to react. The efficiency of labelling depends on the reaction speed and on the reaction time. Its value was determined before labelling (see section 3.3.). The average value of 70% was considered satisfactory and therefore there was no reason to extend the reaction time. The efficiency was not affected by slight modification of the FITC concentration as shown above.
4. 4. The desired degree o f labelling 1 mol of FITC/mol of insulin was regarded as the optimum ratio for labelling. Although free amino groups do not play a considerable role in the biological activity of insulin (Titze, et al., 1962) a higher ratio is however not recommended. The weight ratio of the dye is 1 0 - 2 0 times higher, compared with other proteins, and this is enough for visualization of the immunofluorescent reaction.
4.5. Purification o f the conjugate The unbound dye was removed by gel filtration on a Sephadex G-25 column. Elution was carried out with carbonate buffer (pH 9.0) because the insulin precipitated in buffered saline. The loss of protein was similar to that obtained with other proteins and probably could have been reduced by using Sephadex G-5 or G-10.
4. 6. Chemical and biological characterization o f the conjugate The FITC-insulin conjugates were subjected to chemical analysis. Dye content, protein content, dye-protein weight- and molecular ratios, fading and biological characteristics were examined. The methods and the results obtained are published in separate papers (Jobb~gy and Jobb~gy, 1973a, b).
REFERENCES Berns, A.W. and H.T. Blumenthal, 1962, J. Lab. Clin. Med. 60, 535. Berns, A.W., T.T. Oweno, Y. Hirata and H.T. Blumenthal, 1962, Diabetes 11,308.
382
A. JOBBAGY and G.M. JOBBAGY
Jobb~igy, A.R. and G.M. Jobb~igy, 1973a, J. Immunol. Methods 2, 159. Jobb~gy, A.R. and G.M. Jobb~igy, 1973b~ J. Immunol. Methods 2, 169. Jobb~gy, A.R. and G.M. Jobb~gy, 1973c, J. lmmunol. Methods, in press. Parker, J.W., F.R. Elevitch and G.M. Grodsky, 1963, Proc. Soc. Exptl. Biol. Med. 113, 48. Titze, F., G.E. Mortimore and N.R. Lomax, 1962, Biochim. Biophys. Acta 59, 336.
FTC--INSULIN
CONJUGATES.
I. P R E P A R A T I O N OF F L U O R E S C E I N
THIOCARBAMYL-INSULIN CONJUGATES
A. JOBB/kGY and G.M. JOBBAGY Laboratory o f lmmunopathology, Hungarian State Institute o f Dermatology and Venereology, Budapest, Hungary
Received 15 February 1972
Accepted 30 June 1972
Fluorescein thiocarbamyl-insutin conjugates were prepared, partly for the immunofluorescent technique, partly to study the labelling process. The quality of the raw materials and the characteristics of the FITC-insulin reaction were investigated. Labelling was carried out with a calculated amount of FITC. This technique enabled us to control the labelling process and to obtain conjugates with most favourable characteristics.
1. INTRODUCTION Fluorescein t h i o c a r b a m y l - i n s u l i n conjugates are used to demonstrate intracellular insulin antibodies in patients with diabetes mellitus (Berns and Blumenthal, 1962; Berns et al., 1962; Titze et al., 1962; Parker et al., 1963). Since antibodies develop after treatment with bovine or swine insulin, a mixture containing both insulin preparations was labelled with fluorescein isothiocyanate (FITC) for use in the tests. Labelling was carried out with crystalline FITC isomer I. The characteristics of the FITC preparation were studied in order to obtain information about the dye and isothiocyanate content. These data made it possible to ensure the most favourable degree o f labelling. Theoretically i mol o f FITC is required to label 1 mol of insulin. In practice, however, a little more dye is needed because of the incomplete homogeneity of the labelling process. Ratios from 1 - 1 . 5 may be regarded as most favourable, with less influence on the biological activity o f insulin than higher values. In this paper a m e t h o d is suggested for the preparation o f F T C - i n s u l i n conjugates, with o p t i m u m labelling efficiency.
372
A. JOBB,~GY and G.M. JOBBA,GY
2. MATERIALS AND METHODS
2.1. lnsulin A mixture of porcine (mol.wt. 5777) and bovine (mol.wt. 5733) preparations, was obtained from Kt3bhnyai Gydgyszer~rugy~r (Budapest); for the calculations an average mol.wt, of 5755 was employed. The preparation had been recrystallized 10 times and its activity was 23 units/rag.
2.2. Reagents Crystalline fluorescein isothiocyanate isomer I (mol.wt. 389.39) was purchased from SPOFA (Czechoslovakia). 0.5 M carbonate buffer pH 9.0, containing Na2 CO3 (anhydrous) 1.2 g; NaHCO3 7.4 g; distilled water to 200 ml; a 1 : 10 dilution was used for gel filtration.
2.3. Determination of the dye content of conjugates Dye content was determined by direct measurement of optical density at 496 nm in 0.1 N NaOH (Jobb~gy and Jobb~igy, 1973a, b).
2.4. Protein determittation of conjugates The protein determination was carried out according to the Biuret method. Extinction was measured at 565 nm and the value obtained corrected for absorption by the dye at tile same wavelength.
3. EXPERIMENTS Three samples of insulin were labelled in parallel with FITC. The volume of each conjugate was 5 ml and the protein content 15 mg/ml; their expected respective molecular ratios FTC : insulin were: 1.05, 1.22 and 1.40 : 1. To ensure the desired degree of labelling the following preliminary experiments were carried out.
3.1. Determination of the dye content of the fluoresce& isothiocyanate preparation FITC preparations often contain a certain amount of colourless impurities and/ or preserving agents. It is therefore necessary to determine the total dye content. 5 mg dye was dissolved in 100 ml 0.1 N NaOH. The solution was diluted 1 : 25 and readings were made at 488 nm and 320 nm. A solution containing 1 /ag/ml fluorescein was used as reference standard. Dye content was calculated according to the following formula:
FTC-insulin conjugates. I
cd -
Q'F1TC QFITC
373
FMwl "FMel ' CF" Vd' Dd" EFITC 488 corr. QFITC " EF 489 corr.
•
103
2.40735 • EFITC 488 corr.
(1)
where -- The dye content of the FITC preparation;
ca
= The quantity of the dye determined photometrically, in mg; = The total amount of dye employed for the dye content determination, in mg (5 mg); FMwL = The ratio of the molecular weights of F1TC and F (1.1721); FMe 1 = The ratio of the molecular extinction coefficients of F and FITC (0.961218); CF -- The concentration of the fluorescein standard solution, in/ag/ml (1/ag/ml); -- The volume of the dye solution (100 ml); Dd = The dilution of the dye solution employed for the photometric readings (1 : 25); EFITC 488 corr. = The corrected extinction of the FITC solution at 488 nm wavelength (0.2077); The corrected extinction of fluorescein at 489 nm wavelength EF 489 corr. = (0.234). The dye content of SPOFA FITC isomer I was found to be 0.984. O'FITC QFITC
3. 2. Determination o f the optimum labelling efficiency The dye content gives information about the ratio of the coloured constituents of dye products. This value, however, is not equal to the FITC content, because the side- and degradation products of FITC are also coloured. The true FITC content varies according to the type of product and the duration and condition of storage. The determination of the optimum labelling efficiency is an arbitrary measure which provides an indication of the true FITC content of the dye products. 5 ml albumin (50 mg/ml) was labelled with 1 mg SPOFA FITC at 25°C for 24 hr. The albumin conjugate was gel filtered and the FTC content determined. The ratio bound FTC : added dye (coloured ingredients) expresses the optimum labelling efficiency. CFTC corr. a ' D1 a " Va Col
QFITC " Cd" 103
374
A. JOBB,~GY and G.M. JOBB,~GY FMwl" FMw2" FMe2a ' CF ' D l a ' D2a" Cpa" Va" EFTC 492 corr. Dla" QFITC" Cd" %ca" 103"E F489 con.
(2)
= 2.62577 - EFTC 492 corr. whe re:
= The optimum labelling efficiency; = The dye content of the FTC albumin conjugate, corrected for loss of dye during gel filtration, in/ag/ml; The amount of FITC added (1.0165 rag); QF1TC = The ratio of the molecular weights of FTC radical and FITC FMw2 = (1.0026); The ratio of the molecular extinction coefficients of FITC and FMe2a FTC radical bound to albumin (1.856); The dilution of the FTC-albumin conjugate for photometric O2a = readings applied to the dye content determination (1 : 25); a = The protein content of the albumin solution (50 mg/ml); Va = Volume of the albumin solution (5 ml); The protein content of the FTC-albumin conjugate (21.27 mg]ml); ca EFTC 492 corr. = The corrected extinction of the F T C - albumin conjugate at 492 nm wavelength (0.3455). The optimum labelling efficiency, using SPOFA FITC, was 0.922.
Eo I CFTC corr. a
3. 3. Determination of the efficiency of labelling Three parallel samples of insulin (5 ml-15 mg/ml) were labelled with different amounts of FITC (table 1) at 25°C for 4 hr. Under these circumstances the reaction is usually not complete. The ratio bound FTC : FITC added expresses the efficiency of labelling.
Table 1 Determination of the labelling efficiency of FTC insulin conjugates. Conjugate
FMe2i
EFTC 490 corr. QFITCmg
Cpic mg/ml
El
1
1.1002
0.2725
8.5294
4.1606
0.6836
2
1.1339
0.3410
9.9509
4.3173
0.7283
3
1.0539
0.3970
11.3725
4.2851
0.6948
Average
0.7022
FTC-insulin conjugates. I
375
CFTC corr. i " Vi ' D 3
E1 QF1TC" Cd "Eo I " 103 FMwl" FMw2 " FMel " FMe2i " DI i " D2i " Cpi • Vi " EFTC 490 corr. D1 i" QFITC " Cd " Eo 1 " Cpic" 103 • E F 489 corr.
= 80•9207
FMe2i "EFTC 490 corr.
(3)
QFITC " Cpic where: = Labelling efficiency of the insulin; = The dye content of the FTC-insulin conjugate, corrected for loss of dye during gel fltration; vi = Volume of the insulin solution used for labelling (5 ml); FMe2i = The ratio of the molecular extinctions of FITC and FTC bound to insulin (table 1); D2i = The dilution of the FTC-insulin conjugate used for photometric readings (1 : 200); Cpi = The protein content of the insulin solution used for the labelling, in mg/ml (15 mg/ml); Cpic = The protein content of the insulin conjugate, in mg/ml (table 1); EFTC 490 corr. = The corrected extinction of the FTC-insulin conjugate at 490 nm wavelength (table 1). The labelling efficiencies for insulin were similar in the three conjugates and showed an average o f 70%. In the range 1 - 1 . 5 mol of dye to 1 tool of protein, the efficiency of labelling was not affected by the d y e - p r o t e i n ratio.
El CFTC corr. i
3•4. Calculation o f the desired degree o f labelling Insulin has 3 free amino groups which are able to react with FITC in an additional reaction. Theoretically at least 1 FITC mol is needed for 1 mol of insulin• A higher dye ratio is not recommended, because of its effect on the biological activity of insulin. The labelling is not homogeneous - some insulin molecules bind two FTC radicals and some none at all. To ensure the labelling of all insulin molecules, the dye ratio was increased above 1. The FITC ratios of the three parallel samples were: 1.05, 1.225, 1.40. For the calculation o f the amount of FITC necessary, the desired degree of labelling must be expressed as a weight ratio.
376
A. JOBB,~GY and G.M. JOBB,~GY CFITC
Dtw 1
(4)
Cpi
where: Dlw I = The desired degree of labelling, in terms of a weight ratio; CFITC = The amount of FITC added, in/ag/ml; Cpi = The insulin content of the solution, in mg. So far only the molecular ratio of the desired degree of labelling has been determined; the weight ratio can be calculated from this value by the following formula: CFITC Dlml
=
MWFITC. 10 3-
=
Cpi
CFITC • Mw. l Cpi" MWFITC • 103
(5)
Mw i where: Dim I
= The desired degree of labelling, in terms of a molecular ratio; = The molecular weight of insulin. CFITC in equation (5) is substituted in equation (4).
Mw i
Dim 1 ' Cpi" MWFITC - 10 3 CFITC
Dim 1 • Cpi" MWFITC • 10 3 Dlwl
(6)
Mw i
Cpi" M w i
Dim 1 • M W F I T C
•
103
Mw i
= 67.6612 • Dlm l
(7)
where: MWFITC
= The molecular weight of FITC (389.39); = The molecular weight of insulin (5755). The results are listed in table 2.
Mw i
3. 5. Calculation o f the amount o f F I T C used f o r the labelling o f insulin
Three 5 ml samples of an insulin solution (15 mg/ml) were labelled with FITC. From the above data the necessary amount of dye was calculated according to the following formula:
FTC-insulin conjugates. I
377
_ Cpi" Vi" Dlw1
QFITCi
-
E1 . Eol" Cd" 103
(s)
= 0.12005 - Dtwl
where: QHTCi = The amount of FITC used for the labelling of insulin, in mg. For other symbols see the previous equations. The results are listed in table 2. Table 2 The desired degree of labelling. Conjugate
The desired degree of labelling expressed as: molar ratio Dlml
weight ratio Dlwl
The amount of FITC used for labelling QFITC
1
1.050
71.0442
8.5288
2
1.225
82.8849
9.9503
3
1.400
94.7256
11.3718
3. 6. Labelling o f insulin with FITC 375 mg insulin were dissolved in 10 ml 0.03 N HCL, 10 ml physiological saline and 5 ml 0.5 M Na2 CO3 buffer (pH 9.0) in this order. The final concentration of insulin was 15 mg/ml. 5 ml of this stock solution were placed in a 25 ml Erlenmeyer flask. For each sample a 3 ml FITC solution was prepared with the necessary amount of FITC per ml (table 2). 1 ml was measured with an Autospencer for the labelling o f the insulin, and 1 ml was used for the FMe2i factor determination. The dye solution was added to the insulin solution at room temperature drop by drop, with continuous stirring. The flasks were then put in a Vibrotherm and shaken for 4 l~r at 25°C. Unbound dye was removed on a Sephadex G-25 (fine) column equilibrated with 0.05 M carbonate buffer. The volume of the column was 4 times the volume of the insulin solution. During gel f'dtration part o f the conjugate remained in the gel. This loss of insulin can be calculated from the original protein content (Cpi) and the protein content of the conjugate (Cpic), according to equations 9 and 10. % i loss
= C p i - %ic "D3
% i loss %
_ Cpiloss" 100 Cp i
(9)
(10)
Conjugates
Protein content of conjugates Cpic
4.1606 4.3173 4.2851
Protein content of insulin solution of mg/ml
15
15
15
2.8382
2.9218
3.0887
Dilution of conjugate D3
12.1620
12.6143
12.8508
Protein content related to the original volume Cpic.r.
Table 3 Determination of the protein loss of insulin conjugates.
2.8380
2.3857
2.1492
mg/ml
18.920
15.905
14.328
%
Loss of insulin
1.4
1.225
1.05
Degree of labelling
,<
~a e~
~,, C~
oo
FTC-insulin conjugates. I
379
where: Cpi loss
= The loss of insulin, in mg/ml;
Cpi loss %
= The loss of insulin, in percent.
The protein loss varied from 14-19%, and increased proportionally with the degree of labelling (table 3). The conjugates were characterized chemically and freezedried. The methods used for chemical characterization will be published in a subsequent paper (Jobb~gy and Jobb~gy, 1973c).
4. RESULTS AND DISCUSSION The aim of our experiments was to prepare a reagent for the demonstration of intracellular anti-insulin antibody in patients treated with insulin of various animal origins and to study the labelling reaction. Pure crystalline insulin, being chemically homogeneous, is an ideal substance for investigating the labelling process. In order to obtain a high quality reagent, the following aspects of the process have been studied: 1. The quality of the dye preparation; 2. The characteristics of the protein solution to be labelled; 2. The characteristics of the FITC-protein reaction; 4. The desired degree of labelling.
4.1. The quality o f the dye preparation Manufacturers of dye preparations do not all supply data about the quality of their products. In the majority of cases these are contaminated with coloured and colourless impurities to a greater or lesser extent, depending upon the grade of preparation and the duration and condition of storage. A certain amount of information can be obtained from the following: a. Determination of the dye ratio (coloured : colourless ingredients); b. Determination of the ratio FITC : dye; c. Calculation of the actual FITC content of the preparation. The methods for the determination and the data for SPOFA FITC isomer I are outlined in sections 2 and 3. Several dye preparations were compared. Results and details of the methods have been published (Jobb~gy and Jobb~gy, 1973a,b). Both methods supply useful information on the quality and the purity of dyes. The true FITC content may be calculated by multiplying the two ratios. Even crystalline FITC preparations need chemical characterization, because during their storage the FITC content decreases. Thus, the preparation we employed contained only 90% FITC.
380
A. JOBB~,GY and G.M. JOBB,~GY
4.2. The characteristics o f the prote& solut&n selected for labelling Volume, protein content and biological activity must be determined before labelling. The inorganic contamination of the crystalline insulin preparation was very low, therefore the protein content was not examined.
4. 3. The characteristics o f the FITC protein reaction 4.3.1. Speed of reaction Several factors may have an effect on the reaction speed:
a. Concentration o f insulin An insulin concentration of 15 mg/ml was selected for the labelling, because this value lies within the normal protein concentration range.
b. Concentration o f FITC The concentration of FITC employed was determined by the protein concentration and the desired degree of labelling. The latter was somewhat more than 1 tool dye/1 mol insulin, but the values were close to each other. Therefore, the dye concentration did not play a significant role in the modification of the reaction speed over the range employed.
c. Temperature o f the reaction m&ture 25°C was selected for labelling, although 4°C has been suggested by several authors. At that temperature, however, the reaction speed is very low. For practical and economical reasons - shorter labelling duration, smaller amount of dye - the warm labelling process was chosen.
d. Agitation o f reaction m&ture The Vibrotherm provided a constant temperature and continuous shaking of the reaction mixture. Thus the reaction speed was increased, and one could expect a more homogeneous distribution of the FITC molecules.
e. pH o f the reaction mixture Generally pH 9.0 is used. At a lower pH the reaction speed is very slow, while at higher pH the biological activity of insulin may be decreased.
FTC-insulin conjugates. I
381
4.3.2. Duration of the labelling process 25°C is a favourable temperature for some bacterial growth, therefore a reaction time of 4 hr was employed. 4.3.3. Efficiency of labelling During 4 hr of labelling the reaction is not complete. Under these conditions only a proportion of the dye molecules are able to react. The efficiency of labelling depends on the reaction speed and on the reaction time. Its value was determined before labelling (see section 3.3.). The average value of 70% was considered satisfactory and therefore there was no reason to extend the reaction time. The efficiency was not affected by slight modification of the FITC concentration as shown above.
4. 4. The desired degree o f labelling 1 mol of FITC/mol of insulin was regarded as the optimum ratio for labelling. Although free amino groups do not play a considerable role in the biological activity of insulin (Titze, et al., 1962) a higher ratio is however not recommended. The weight ratio of the dye is 1 0 - 2 0 times higher, compared with other proteins, and this is enough for visualization of the immunofluorescent reaction.
4.5. Purification o f the conjugate The unbound dye was removed by gel filtration on a Sephadex G-25 column. Elution was carried out with carbonate buffer (pH 9.0) because the insulin precipitated in buffered saline. The loss of protein was similar to that obtained with other proteins and probably could have been reduced by using Sephadex G-5 or G-10.
4. 6. Chemical and biological characterization o f the conjugate The FITC-insulin conjugates were subjected to chemical analysis. Dye content, protein content, dye-protein weight- and molecular ratios, fading and biological characteristics were examined. The methods and the results obtained are published in separate papers (Jobb~gy and Jobb~gy, 1973a, b).
REFERENCES Berns, A.W. and H.T. Blumenthal, 1962, J. Lab. Clin. Med. 60, 535. Berns, A.W., T.T. Oweno, Y. Hirata and H.T. Blumenthal, 1962, Diabetes 11,308.
382
A. JOBBAGY and G.M. JOBBAGY
Jobb~igy, A.R. and G.M. Jobb~igy, 1973a, J. Immunol. Methods 2, 159. Jobb~gy, A.R. and G.M. Jobb~igy, 1973b~ J. Immunol. Methods 2, 169. Jobb~gy, A.R. and G.M. Jobb~gy, 1973c, J. lmmunol. Methods, in press. Parker, J.W., F.R. Elevitch and G.M. Grodsky, 1963, Proc. Soc. Exptl. Biol. Med. 113, 48. Titze, F., G.E. Mortimore and N.R. Lomax, 1962, Biochim. Biophys. Acta 59, 336.