Immunofluorescence reagents, and the method for their preparation

New immunofluorescence reagents prepared by reacting together a protein, a cross-linking agent and a dye, which are of general formula ##STR1## and which fluoresce between 600 and 900 nm.

In clinical diagnosis the quantitative determination of small compounds 
such as hormones, drugs or metabolytes by immunological methods is 
extremely important. One of the methods for quantifying immunoreactions is 
to mark the antigens and antibodies with fluorescent chromophores such as 
fluorescein or rhodamine isothiocyanate. The main problem connected with 
the use of this method is the inherent fluorescence of the biological 
samples. In this respect, many substances such as the flavins, pyridine 
coenzymes and serum proteins fluoresce intensely in those regions of the 
spectrum in which the fluorochromes normally used as probes also emit. The 
use of probes which fluoresce in the red-near infrared region (600-900 
nm), and thus fairly distant from the inherent fluorescence of the samples 
under examination (300-450 nm), would enable the background due to natural 
substances to be eliminated, and the sensitivity of the method to be 
increased. Many dyes which are free in solution are known to have these 
spectral properties, even though their intensity of fluorescence is fairly 
weak, but what is not obvious is that they maintain these properties if 
their structure is altered in some way by a covalent bond with another 
molecule. 
This result has now been obtained, and forms the subject matter of the 
invention, by covalently bonding dyes which fluoresce in the near-infrared 
region to various proteins by using bifunctional reagents which react with 
--NH.sub.2 groups of the fluorochromes, without these dyes losing their 
spectural characteristics and in fact improving them by increasing 
fluorescence by 2 to 4 times (see FIG. 1 and FIG. 2). 
FIG. 1 shows the emission spectra of an anti .gamma. immunoglobulin-Nile 
blue conjugate (curve 2) and a Nile blue solution (curve 1). 
The chromophore concentration is 2.times.10.sup.-6 M. The excitation 
wavelength is 600 nm. 
The spectra were determined by a Perkin-Elmer MPF-2A spectrofluorometer 
fitted with a HAMAMATSU R446 photomultiplier. The abscissa axis represents 
the wavelength in nm, and the ordinate axis represents the fluorescence in 
arbitrary units. FIG. 2 shows the emission spectra of Protein A--Nile blue 
(curve 3) and HCS--Nile blue (curve 2) conjugates, and free Nile blue 
(curve 1), obtained by excitation at .lambda.=515 nm with an Argon laser. 
The chromophore concentration is 6.times.10.sup.-6 M. The abscissa axis 
represents the wavelength in nm, and the ordinate axis the fluorescence in 
arbitrary units. 
Generally, the bifunctional reagent, represented by the crosslinking agent, 
reacts directly both with the protein and with the dye to form complexes 
of general formula 
EQU [Dye].sub.n L.sub.m Protein 
If this is a carbodiimide, then because these reagents act by activating 
the carboxyl groups to give rise to a carbamido bond between the carboxyl 
groups and the amino group of the dye, complexes are obtained of general 
formula 
##STR2## 
The protein part of these conjungates also maintains its biological 
characteristics, for example immunological if an antigen or antibody. 
It should be noted that the use of these immunofluorescence reagents 
presents no particular difficulties in terms of instruments because it is 
necessary only to fit an infrared-sensitive photomultiplier to a normal 
spectrofluorometer. Measurements of particular sensitivity can be made by 
exciting the fluorochrome with a continuous or pulsed laser of red light 
or near-infrared, such as a helium-neon laser, a ruby laser or more easily 
a dye laser. 
The use of these conjugates, in particular fluorescent antibodies, in 
microscopy presents no difficulty because only an infrared-sensitive film 
may be necessary. It also offers the advantage of being able to 
discriminate in the same microscopic preparation with respect to zones 
coloured with antibodies marked with conventional fluorochromes which 
fluoresce in the green-yellow region of the spectrum. The present 
invention therefore relates to new immunofluorescence reagents of general 
formula 
##STR3## 
in which n is a whole number from 1 to 4 
m is a whole number equal to n, except when the cross-linking reagent is a 
carbodiimide, in which case m=0. 
P is a protein 
L is a cross-linking agent residue 
X is an oxygen or sulphur atom 
y is a group chosen from NH, NH.sub.2, NR', NR'R" where R' and R", which 
may be the same or different, are alkyl, aryl, arylalkyl or alkylaryl 
radicals, possibly substituted, or an OCOR' group where R' has the 
aforesaid meaning 
R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.7, R.sub.8, which may be 
the same or different, are either a hydrogen atom or an amino group or an 
alkyl, aryl, alkylaryl or arylalkyl radical, possibly substituted; R.sub.1 
and R.sub.2 and/or R.sub.7 and R.sub.8 can also be bivalent radicals which 
close together to thus form a condensation ring with the cycloaromatic. 
At least one of these (most frequently R.sub.3) must be an amino group 
which on reacting with the cross-linking reagent gives rise to a --NH--L 
or --N.dbd.L--group. 
These reagents are prepared by reacting together at least one compound 
chosen from each of the following classes 
(a) a protein 
(b) a dye chosen from those oxazines or thiazines having an absorption in 
the visible range with .lambda. max between 550 and 800 nm, and having at 
least one free NH.sub.2 and a fluorescence with emission between 600 and 
900 nm 
(c) a cross-linking agent, generally bifunctional reagents of which the 
groups are able to react with the amino group of the dye. 
Some compounds are listed hereinafter by way of example which can form part 
of the aforesaid classes. 
Protein 
(i) Protein A from Staphylococcous aureus 
(ii) Protein hormone 
(iii) .gamma.-immunoglobulin (Ig) of the various classes IgG, IgM, IgA, 
IgD, IgE. 
Dye 
(i) Nile blue (C.I. 51180) 
##STR4## 
(ii) Cresyl violet 
##STR5## 
(iii) Toluidine blue (C.I. 52040) 
##STR6## 
(iv) Brilliant cresyl blue (C.I. 51010) 
Cross-linking agent 
(i) diisocyanate of general formula O.dbd.C.dbd.N--R--N.dbd.C.dbd.O where R 
is an alkyl, aryl, alkylaryl or arylalkyl radical, possibly substituted 
(ii) carbodiimide of general formula R--N.dbd.C.dbd.N--R' where R, R' are 
alkyl, aryl, alkylaryl or arylalkyl radicals, possibly substituted, and 
can be the same or different 
(iii) dialdehydes of general formula CHO--R--CHO where R is an alkyl, aryl, 
alkylaryl or arylalkyl radical, possibly substituted 
(iv) ethylene-maleic anhydride copolymers (EMA) of general formula 
##STR7## 
(v) dinitrobenzenes of general formula 
##STR8## 
The reaction between the three compounds can either be carried out starting 
from the substrates alone, or can be carried out in the presence of a 
medium compatible with the components, for example water, aqueous 
solutions of one or more salts, or buffer mixtures. The pH of the medium 
can vary from 4.5 to 9.5, and in addition small quantities of organic 
solvents can be added in order to increase the solubility of the dye. The 
reaction temperature varies around ambient temperature (from 
4.degree.-5.degree. C. to the protein denaturation temperature). 
The reaction is generally carried out with a large excess of dye over the 
protein, in order to prevent protein-protein reactions, this excess 
varying from 4 to 50 times in terms of molar concentration. 
The reaction is also carried out with a large excess of crosslinking agent 
over the total quantity of protein plus dye.

All operating details will be apparent from the following examples. 
These examples do not however limit the invention, which can be 
advantageously applied to any three substrates. 
EXAMPLE 1 
The immunoglobulin fraction of an anti human immunoglobulin G rabbit serum 
(anti h IgG) was marked both with Nile blue (CI 51180) and with toluidine 
blue (CI 52040), using glutaraldehyde as the bifunctional reagent. 
Glutaraldehyde is added to a solution containing 2 mg of anti h IgG and 
1.3.times.10.sup.-4 mmoles of Nile blue or toluidine blue in 1 ml of 
potassium phosphate buffer (0.01M pH 7.1) until the final glutaraldehyde 
concentration is 0.05%. 
The reaction is allowed to proceed for 30 minutes under stirring at ambient 
temperature, and is then blocked by trapping the free aldehyde groups with 
sodium bisulphite. The unreacted dye is eliminated by chromatography over 
Sephadex G25. 
In a typical experiment, a fluorescent anti h IgG--Nile blue conjugate is 
obtained with a molar protein:dye ratio of 1:1.8, or an anti h 
IgG--toluidine blue conjugate with a molar ratio of 1:1.2. 
The dye concentration was calculated using for the Nile blue a molar 
extinction coefficient of 5.8.times.10.sup.4 at .lambda.=635 nm, and for 
the toluidine blue a molar extinction coefficient of 3.1.times.10.sup.4 at 
.lambda.=620 nm. 
The protein concentration was calculated using a .epsilon..sup.% =14 at 
.lambda.=280 nm, and correcting for dye absorption in the ultraviolet, or 
by determining the nitrogen with the Kjeldahl micromethod. 
Both the conjugates significantly maintain the antibody property of bonding 
human immunoglobulins. 
EXAMPLE 2 
A conjugate between human chorionic somatomammotropin (HCS) and a dye of 
the oxazine series, namely brilliant cresyl blue (CI 51010), is prepared 
using glutaraldehyde as the reagent. Glutaraldehyde is added to 1 ml of 
sodium phosphate buffer at pH 7.8, 0.05 M containing 2.5 mg of HCS and 
5.times.10.sup.-4 mmoles of dye, until the final glutaraldehyde 
concentration is 0.1%. After 1 hour of reaction at ambient temperature, 
the solution is treated as in example 1 to obtain a conjugate with a molar 
HCS:brilliant cresyl blue ratio of 1:1. 
The ratio was calculated using for the dye a molar extinction coefficient 
of 16500 at .lambda.=633, and for the HCS a .epsilon..sup.% =8.3 at 
.lambda.=278. 
EXAMPLE 3 
Using glutaraldehyde as the cross-linking agent, then under the same 
experimental conditions as example 1 a conjugate is prepared between Nile 
blue and Protein A from Staphylococcus aureus. 
The molar protein-dye ratio of this conjugate is 1:1.5. In the case, when 
not using the Kjeldahl micromethod, a .epsilon..sup.% =1.65 at 
.lambda.=275 was used for determining the Protein A. 
EXAMPLE 4 
Nile blue (CI 51180) is used for marking anti human immunoglobulin (anti h 
IgG), using as the bifunctional reagent toluylene-2,4-diisocyanate (TDIC), 
of which the structural formula is 
##STR9## 
The reaction proceeds in two steps. In the first, the isocyanate group in 
position 4, which is less sterically committed, reacts with the NH.sub.2 
of the anti hIgG. 
In the second step, Nile blue is added at 37.degree. C. to the anti 
hIgG-TDIC conjugate containing no free TDIC, so that the second isocyanate 
group reacts with the amino group of the dye. 
Step 1 
0.05 ml of TDIC are added to a solution of 5 mg of .gamma.-immunoglobulin 
in potassium phosphate buffer of pH 7.5, 0.05M, cooled to 0.degree. C. The 
reaction is allowed to proceed for about 30 minutes at 0.degree. C. under 
vigorous stirring, the mixture then being centrifuged to remove the 
unreacted diisocyanate which precipitates in aqueous solutions at 
0.degree. C. The supernatant is allowed to incubate again at 0.degree. C. 
for a further hour, in order to enable any dissolved TDIC to react. 
Step 2 
0.1 ml of phosphate buffer containing 10.sup.-4 mmoles of Nile blue are 
added to the solution of step 1. The resultant solution is kept at 
37.degree. C. for 1 hour under stirring, and is then dialysed against 0.1M 
ammonium carbonate in order to destroy any unreacted isocyanate group. The 
free dye is removed by dialysis against distilled water or neutral buffer 
followed by chromatography over Sephadex G25. 
The resultant conjugate has a molar portein:Nile blue ratio of 1:2.4, 
calculated using the methods and extinction coefficients for protein and 
dye given in example 1. 
The fluorescence intensity of this conjugate (see FIG. 1) is much higher 
than that obtained on exciting the free chromophore at the same 
concentration. 
EXAMPLE 5 
Nile blue (CI. 51180) is conjugated with Protein A from Staphylococcus 
aureus using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (ECDI), of 
which the structural formula is 
EQU CH.sub.3 --CH.sub.2 --N.dbd.C.dbd.N--CH.sub.2 --CH.sub.2 
--CH(NH--CH.sub.3).sub.2. 
50 mg of ECDI are added to a solution containing 5 mg of Protein A and an 
excess of Nile blue to the extent of about ten times molar in 1.2 ml of 
distilled water. The reaction is allowd to proceed under stirring for 
seven hours at ambient temperature, while maintaining the pH between 6 and 
6.8 by adding dilute HCl, after which the product is dialysed against 4-5 
water changes over 48 hours. The colloidal suspension which can form in 
the dialysis bag is eliminated by centrifuging, and any free dye still in 
solution is removed by chromatography over Sephadex G25. 
The Nile blue concentration in the conjugate is determined using a molar 
extinction coefficient in water of 5.8.times.10.sup.4 at .lambda.=635. The 
protein concentration is calculated using a .epsilon..sup.% =1.65 at 
.lambda.=275 nm and correcting for the dye absorption in the ultraviolet 
or, in a more precise manner, by determining the nitrogen with the 
Kjeldahl micromethod. In a typical reaction, a conjugate is obtained with 
a molar Protein A: Nile blue ratio of 1:1.div.1.5 (this ratio can be 
varied by varying the reaction time and temperature) and a fluorescence 
intensity at .lambda.=680 nm which is greater than that obtained by 
exciting the free chromophore at the same concentration. The Protein A 
marked in this manner maintains its property of bonding immunoglobulins, 
as demonstrated by the passive hemoagglutination test, comprising 
agglutination of ram erythrocytes sensitised with rabbit G immunoglobulins 
by Protein A--Nile blue. 
EXAMPLE 6 
A protein hormone, namely human chorionic somatomammotropin (HCS) is marked 
with Nile blue in the presence of ECDI using the procedure illustrated in 
example 5, to which reference should be made for details. 
The reagent concentrations are 2.5 mg of HCS, 10.sup.-3 mmoles of Nile blue 
dissolved in 0.05 ml of ethanol, and 45 mg of ECDI, all in 2 ml of water. 
The protein concentration is determined using a .epsilon..sup.% =8.3 at 
.lambda.=278. 
The fluorescent conjugate obtained has a molar HCS:dye ratio of 1:1, and 
the hormone maintains its antigen properties when incubated with an anti 
HCS antiserum. 
EXAMPLE 7 
Toluidine blue (CI 52040) is used to mark rabbit .gamma.-immunoglobulins 
using ECDI. 
The reaction conditions are as follows. 
The reaction mixture, consisting of 3 mg of .gamma.-immunoglobulins and 
2.times.10.sup.-4 mmoles of toluidine blue in 2 ml of water is adjusted to 
pH 5.6 by dilute HCl. 25 mg of ECDI are added, and the reaction is allowed 
to proceed under stirring at ambient temperature. After about 2 hours, a 
further 25 mg of ECDI are added, and the reaction is allowed to proceed 
for a further 2 hours. 
The solution is finally dialysed against water or a neutral buffer of low 
ionic force for 48-72 hours with various changes. Using a molar extinction 
coefficient of 3.1.times.10.sup.4 for the dye at .lambda.=620 nm, and a 
.epsilon..sup.% =14 for the protein at .lambda.=280 nm, a molar protein: 
toluidine blue ratio of 1:1.1 is calculated for the prepared conjugate. 
The .gamma.-immunoglobulin marked in this manner maintains its 
immunological properties in the presence of anti .gamma.-immunoglobulin.