Protein assay kit

The invention provides a kit for use in assaying protein, which comprises: PA1 an alkaline copper solution containing a tartrate; and PA1 a solution of Folin reagent. An alkaline copper solution containing tartrate has surprisingly been found to remain stable for several months. This allows a kit to be provided for use in a method of protein assay, in which the alkaline copper solution is provided ready-made up for immediate use in the method.

FIELD OF THE INVENTION 
This invention relates to a kit for use in assaying protein. 
BACKGROUND TO THE INVENTION 
There are several methods for the determination of protein quantity in 
samples. These include the use of colour-changing dyes, such as Orange G, 
Bromo cresol green, Pyrocatechol Violet-Molybdenum complex, etc. These 
dyes, when bound with protein, change colour proportional to the amount of 
protein present in the samples. Such assay methods are generally not very 
sensitive. A more sensitive dye-binding technique using Coomassie 
Brilliant Blue G-200 is adversely affected by the presence of detergents 
in a sample and also suffers from wide protein-to-protein variation 
(Bradford, M., Anal. Biochem., 72 248-254, 1976 and U.S. Pat. No. 
4,023,933). 
A variety of turbidimetric methods are also known in which protein is 
precipitated by various agents. These methods also suffer from lack of 
sensitivity and specificity and interference with detergents. 
The most widely used procedure for protein determination involves the 
well-known reaction of protein in alkaline medium with cupric ions 
yielding highly reactive cuprous ions. A method using alkaline copper was 
first developed by Lowry et al (Lowry, Oh. H., Rosebrough, N. J., Farr, A. 
L., and Randall, R. J., J. Biol. Chem. 193, 265-275, 1951) ("the Lowry 
method") in which protein reacted with buffered alkaline copper was 
coupled with Folin phenol reagent (phosphomolybdic/phosphotungstic acid), 
hereinafter referred to as Folin. It is believed that protein reacts with 
alkaline copper and produces cuprous ions and this, in turn, reduces the 
Folin to the characteristic blue reaction colour. 
The Lowry method suffers from many disadvantages. The most serious 
disadvantage is the rigidity of the method. The Lowry method requires 
precisely-timed additions of reagent, immediate vortexing and prolonged 
incubation. Furthermore, the Lowry method also suffers from poor 
reproducibility and interference from a number of commonly used laboratory 
agents. Attempts to simplify the Lowry method have not, so far, been 
successful. Consequently, a need exists for a more flexible and rapid 
method for determination of protein. 
In a recent modification, Smith et al (Anal. Biochem. 150, 76-85, 1985) 
combined the reaction of protein with alkaline copper with bicinchoninic 
acid. Although Smith et al's method has several advantages over the Lowry 
method, it suffers from lack of end-point in the reaction. The colour 
yield of the reaction continues to increase at a rate of 2-3% every ten 
minutes. Consequently, this method is not very accurate and the problem is 
compounded if a large number of samples are analysed in a single batch. In 
addition, Smith et al's method, using bicinchoninic acid, is a slow 
reaction requiring heating and a prolonged incubation period, which makes 
the method time-consuming. 
STATEMENT OF THE INVENTION 
According to the present invention there is provided a kit for use in 
assaying protein, the kit comprising: 
an alkaline copper solution containing a tartrate; and 
a solution of Folin reagent. 
It has now been surprisingly discovered that unbuffered alkaline copper 
solution containing tartrate may be stored for several months without 
significant precipitation or deterioration. This is directly contrary to 
previous thinking, which has always held that an alkaline copper solution 
has an extremely short shelf life. The instability of such a solution has 
meant that it has always had to be prepared in situ, immediately prior to 
use, by combining a copper solution with an alkaline solution. 
Thus, kits for performing protein assays which involve the use of an 
alkaline copper solution have always in the past comprised three separate 
components, ie an alkaline solution, a copper solution and a Folin 
solution. It has never before been thought possible to provide a 
ready-made-up alkaline copper solution for use in a method of protein 
assay. 
It is thought that the presence of the tartrate increases the stability of 
the alkaline copper solution. The tartrate is preferably either sodium or 
potassium tartrate. 
Preferably, the concentration of alkali in the alkaline copper solution is 
at least 0.2N, more preferably from 0.2 to 2N, and most preferably from 
0.4 to 1N. 
Such a kit could be used, for instance, in a method of assaying protein 
which comprises the following steps: 
(a) contacting together a protein-containing solution and the alkaline 
copper solution of the kit; 
(b) contacting together the product of step (a) above and the solution of 
Folin reagent, the amount of the Folin reagent preferably being such that 
the initial pH of the resultant solution is from 11 to 12; and 
(c) allowing the products of step (b) above to incubate at ambient 
temperature until the optical density of the solution reaches a maximum 
value and reading this maximum optical density in order to determine the 
amount of protein in the protein-containing solution. 
Such a method is a two-step procedure in which no incubation period is 
required after the performance of the first step. As far as the second 
step is concerned, maximum optical density may be reached in as little as 
10 minutes and this maximum optical density may be maintained over a 
period of, for instance, 10 to 40 minutes during which the optical density 
measurement may be taken. In the Lowry method, it is necessary to incubate 
for at least 10 minutes the products of the first step of the method and 
then wait for at least 30 or 40 minutes before making the optical density 
measurement after the second step of the method. 
It had previously been considered that the second step of the Lowry method 
had to be carried out at a pH of about 10 and that the reaction solution 
should be maintained at this pH rather than being allowed to move 
downwardly, which would otherwise happen as the reaction proceeds and 
promotes decomposition of the coloured material in the solution. Contrary 
to accepted practice for very many years, it has now been discovered that 
a quite different approach to the pH of the reaction solution enables 
highly reproducible results to be obtained much more quickly than with the 
traditional Lowry method. The key to the discovery is that, in step (b) of 
the method, relatively very rapid reaction takes place at a pH of between 
11 and 12, preferably between 11.4 and 11.9, more preferably between 11.5 
and 11.8 and most preferably between 11.6 and 11.8. At the same time, the 
pH may be allowed to move downwardly rather than being maintained at this 
relatively high level. Movement of the pH downwardly means that the 
coloured species produced are relatively stable. Accordingly, a maximum 
optical density can be reached rapidly and held at the maximum level for a 
considerable period, more than sufficient to allow optical density 
measurements to be taken. 
A relatively high alkaline concentration is preferably used in the kit of 
the invention, and hence in Step (a) of the above reaction. This has the 
added advantage that Step (a) of the reaction proceeds much more quickly 
than the first step of the conventional Lowry method. As a result, the 
normal incubation period which is required with the traditional Lowry 
method is not necessary in the practice of the method of the present 
invention. The Step (a) reactants may be mixed together and then 
immediately the Folin reagent may be added to enable the Step (b) reaction 
to proceed. The amount of alkali to be used has been defined above in 
terms of the concentration of alkali in the copper-containing solution. 
Although, in Step (a) of the method, the copper-containing solution is 
diluted by mixing with the protein-containing solution, in practice this 
does not result in a significant dilution of the alkali since the volume 
of protein-containing solution is normally no more than about one fifth, 
and often considerably less, than that of the copper containing solution. 
It should be appreciated that the use of a relatively large amount of 
alkali in Step (a) of a method of protein assay (as when using the kit of 
the present invention) means that a correspondingly high relative amount 
of Folin reagent is used in Step (b). This ensures that most of the 
cuprous ions released as a result of the reaction between the copper 
(cupric) containing solution and the protein are immediately completed or 
otherwise reacted with the Folin reagent. It is believed that initially a 
colourless product is produced which rearranges to form the coloured 
species. 
Sodium dodecyl sulphate (SDS) may be used in a protein assay method in 
order to counter the influence of nonionic and cationic detergents on the 
assay. For instance, the protein solution may be treated with SDS prior to 
the addition of Folin. Thus, the kit of the invention may additionally 
comprise a solution of sodium dodecyl sulphate. Alternatively, the 
alkaline copper solution may contain SDS. 
The solution of Folin reagent in the kit is preferably in diluted form 
commercially available Folin phenol reagent is usually 2N. In the kit, the 
Folin is preferably in the form of Folin (2N) diluted 4-40 fold 
(0.5N--0.05N or 25%--2.5% Folin solution), more preferably in the form of 
Folin (2N) diluted around 20 fold (0.1N or 5% Folin solution). It has been 
found that such diluted Folin is usable for 6-12 months and longer. 
Therefore, it may be recommended to have a usable shelf life of 6-12 
months or longer. 
It has been found that such a diluted Folin solution, preferably diluted 
around 20 fold, overcomes interference by detergents such as Triton-X100, 
Tween-20, Bri-35 and others when used in protein assay methods. Thus, when 
used in the kit of the present invention, it can eliminate the need to 
include SDS in the alkaline copper solution in order to overcome 
interference by some detergents. 
The kit of the invention is preferably provided with instructions for 
performing a method of protein assay using the kit. These instructions 
preferably relate to a method as described above. The reagents are 
preferably provided in containers of polymerised hydrocarbon, eg in 
polypropylene containers. The Folin is preferably supplied in a diluted 
ready-for-use form.

DETAILED DESCRIPTION 
A typical protein estimation method based on the use of Folin reagent has 
two constituent reagent solutions, the first of which consists of copper 
in a solution of sodium hydroxide (alkali) and sodium or potassium 
tartrate, possibly buffered with sodium carbonate (hereinafter referred to 
as buffered alkaline copper solution), and the second of which is a 
solution of Folin phenol reagent. These reagent solutions have in the past 
always been reported to have short shelf-lives and have therefore always 
been made fresh, immediately prior to use. 
The preparation of the buffered alkaline copper solution involved making 
two separate solutions and mixing them just prior to use. The solutions 
were: 
Reagent-A, containing 2% sodium carbonate in 0.1N sodium hydroxide; and 
Reagent-B, containing 0.5% copper sulphate pentahydrate in 1% sodium or 
potassium tartrate. 
Reagent-A has always been made fresh, since the solution tends to develop 
precipitate and solid residue in storage. Reagent-B is also typically made 
fresh by mixing equal volumes of 1% copper sulphate and 2% sodium or 
potassium tartrate solutions. The working buffered alkaline copper 
solution would typically be made by mixing 50 parts of Reagent-A and 1 
part of Reagent-B. 
However, it has now been discovered that unbuffered alkaline copper 
solution, i.e. alkaline copper solution without a sodium carbonate 
buffering agent, can be stored for several months without significant 
precipitation and deterioration, provided tartrate is present (see Example 
11 below). It is therefore possible to prepare a long term storable 
alkaline copper solution by mixing an alkaline solution containing 
tartrate with a copper solution. SDS may also be added to the alkaline 
copper solution. Thus, a kit in accordance with the invention comprises a 
ready-made alkaline copper solution, which may be stored prior to use in a 
protein assay method. 
The Folin phenol reagent has always in the past been made fresh from 2N 
concentrated Folin solution, any unused diluted solution being discarded. 
However, it has now been discovered that dilute Folin phenol reagent (ie 
between about 0.5 and 0.05N) can be stored for several months at room 
temperature. The kit of the invention preferably comprises just such a 
ready-diluted Folin solution. It is important for long-term storage that 
pure de-ionised water is used in the preparation of a dilute solution of 
Folin reagent, and that the diluted Folin solution is protected from 
light. The diluted Folin solution should be stored in a container made of 
a polymerised hydrocarbon, such as polypropylene. 
For estimation of the amount of protein in a sample, the sample is first 
treated with alkaline copper solution. It is widely believed that cupric 
copper in an alkaline medium reacts with protein and forms a 
copper-protein complex which in turn releases cuprous ions. 
It has been discovered that when protein is treated with an alkaline copper 
solution containing a high concentration of alkali, the reaction of copper 
is almost instantaneous and requires no incubation for the subsequent 
reaction steps. In the experiments described below, in which protein was 
treated with alkaline copper solution containing 0.4N and 1N sodium 
hydroxide, the reaction of alkaline copper with protein was almost 
instantaneous and required no incubation. Example 5 below clearly proves 
that the reaction of copper with protein was so rapid that in 15 seconds 
(the time it takes to vortex the mixture to achieve a uniform mixing of 
the reagents with protein), the reaction of alkaline copper with protein 
was complete, and the colour yield of the reaction was identical to the 
control test sample which was incubated for 20 minutes in alkaline copper. 
Folin reagent is next introduced into the copper-treated protein solution, 
which results in a characteristic blue colour. Many methods using Folin 
reagent for the estimation of protein recommend the addition of an amount 
of Folin reagent into the copper-treated protein which would give a 
reaction mixture of approximately pH 10. The addition of Folin reagent is 
followed by at least a 30 minute incubation period. Reaction at pH 10 is 
recommended to give a maximum yield of reaction colour and a greater 
stability to reaction colour. 
However, a protein assay reaction between pH 10 and 11 has several 
disadvantages: 
at pH 10, the reaction progresses slowly and takes a long period of 
incubation to reach its maximum value; 
30 minutes' incubation does not bring the reaction to its end-point; and 
the optical density of the reaction colour continues to increase for a long 
time after 30 minutes' incubation, at a rate of 5% to 25% per hour, 
depending on the starting pH of the reaction mixture. The increase in 
optical density contributes to error in the protein estimation. 
Protein estimation can, however, be performed more rapidly and with 
substantially increased sensitivity and reproducibility at high alkaline 
pH, i.e. between pH 11 and 12. It has also been found that the rapid 
release of reaction colour between pH 11 and 12 produces results which are 
more reproducible than slow release, as shown in FIG. 8. At pH 11-12 the 
reduction and colour development of Folin reagent with copper-treated 
protein reaches a maximum and an end-point more rapidly and, in addition, 
the reaction produces a plateau of stable optical density for the 
reaction. The higher the pH, the more rapidly colour development takes 
place. It has also been found that at the preferred pH of between 11.5 and 
11.9, the colour development reaches a maximum and an end-point within 10 
to 15 minutes, and stays nearly constant. This enables reliable 
determination of a large number of samples in a batch. It has also been 
found that after reaching the maximum, the decrease in optical density in 
one hour could be as little as 1% to 2% when measured in steady state, as 
shown in FIG. 3. 
Thus, the components of a kit in accordance with the invention should 
ideally be reacted together in such relative proportions as to yield an 
initial pH, in the copper solution-protein mixture, of the order described 
above. 
It has also been found possible to perform a protein assay within a 
pre-selected time by pre-selecting the reaction commencing pH of the assay 
mixture and reading the reaction colour at the plateau of the maximum 
optical density. The pre-selection of assay time is made possible by 
carefully selecting the amounts of alkaline copper solution and Folin 
solution which, on mixing, could result in a pH at which the reaction 
colour will reach the maximum and end-point within the pre-selected time. 
Tables 1, 2 and 3 in Example 3 give the reaction commencing pH required 
for various pre-selected times, and the length of time for which the 
optical density remains nearly constant. 
It has been discovered that at the reaction commencing pH in buffered 
alkaline medium around pH 11.70, the reduction of Folin and colour 
development reaches maximum in around 10 minutes, and stays nearly 
constant for 15 to 20 minutes, which is sufficient to allow reading of 
40-50 separate samples, and in 40 minutes after reaching the end-point 
maximum, the colour drift is within 3% to 4%. The optical density drift of 
3% to 4% is comparable to the drift in the optical density reported for 
the widely used method of protein determination by Bradford, M. M. (Anal. 
Biochem. 1976, 72, 248-254), and considerably better than the 16% drift in 
the optical density of another widely used method by Smith, P. K. et al 
(Anal. Biochem., 1985, 150, 76-85). In addition, it has also been found 
that, in a real determination in accordance with the present invention, 
the expected drift was not detected, as is shown in Example 3.1 and FIG. 
8. 
A further improvement in the reaction is achieved when a kit containing an 
unbuffered alkaline copper solution is used for the assay. In unbuffered 
alkaline medium, the pH of the reaction drops unhindered and more rapidly, 
and in less than 10 minutes the pH drops as much as 0.15-0.6 units for a 
reaction commencing between pH 11 and 12 (Table 2, FIG. 3). This finding 
has two advantages: 
it allows a rapid release of reaction colour at a very high alkaline pH; 
and 
as soon as the maximum reaction colour is achieved and before substantial 
decomposition of colour can begin, the pH of the reaction also shifts to a 
pH at which the decomposition of the reaction colour is very considerably 
reduced (Tables 1 and 2, FIG. 3). 
The reaction shown in FIG. 3, which could be carried out using a kit in 
accordance with the invention, commences at a pH around 11.70. In less 
than 10 minutes the release of colour reaches a maximum value and 
concurrently the pH of the reaction mixture drops by 0.2 units to pH 11.5. 
At pH 11.5, the optical density of reaction colour is virtually unchanged 
for over 30 minutes (Tables 1 and 2, FIG. 3) and in the next 30 minutes, 
the optical density drops by a mere 2% to 3%. The shift in optical density 
of 2% to 3% in the second half-hour of a one-hour period is less than the 
method of Bradford cited above. 
In a further study, neither sodium carbonate nor sodium tartrate were 
present in the alkaline copper solution. The removal of tartrate from the 
alkaline copper solution (Example 3.3) lowered the rate of reduction of 
Folin with copper-treated protein and consequently it took longer for the 
reaction colour to reach a maximum value, and at maximum the reaction 
colour was generally more stable. The results in Table 3 show that a 
reaction commencing at pH 12 took approximately 10-12 minutes to reach the 
maximum as compared to 5 minutes in unbuffered and buffered alkaline 
solution (Tables 1 and 2). In addition, in the absence of tartrate, the 
reaction colour was more stable after reaching the maximum value, as shown 
in Table 3. 
However, it has been found that tartrate improves the stability of an 
alkaline copper solution, whereas in the absence of tartrate copper tends 
to precipitate from the solution. The higher the concentration of tartrate 
in fact, the more stable the alkaline copper solution. 
It has been discovered that when the tartrate concentration in an alkaline 
copper solution is around 0.1% or three times (.times.3) the concentration 
of copper, the alkaline copper solution is stable for months. 
Preferably, the tartrate concentration in the alkaline copper solution 
should be over 0.2% or five times (.times.5) the concentration of copper. 
Most preferably, the concentration of tartrate should be over 0.5% or ten 
times (.times.10) the concentration of the copper (see Table 4). 
When Folin is added to a copper-treated protein, maximum colour results if 
the reduction occurs at a pH around pH 10. The reduction of Folin at 
higher pH between pH 11-12 results in lowering of the yield of reaction 
colour and consequently reduces the sensitivity of an assay. It has been 
discovered that the colour yield of the reaction at higher alkaline pH 
could be increased to its maximum value or to a level which is comparable 
to the traditional methods based on the use of Folin reagent. The yield of 
reaction colour is maximised by increasing the amount of Folin in an 
assay, which is achieved by increasing the concentration of alkali in the 
alkaline copper solution used. Thus, the kit of the invention includes an 
alkaline copper solution having a relatively high concentration of alkali. 
Example 4 shows that as the concentration of alkali is increased in an 
alkaline copper solution, it requires correspondingly increased amounts of 
Folin to achieve a pH of around 11.7 and consequently the colour yield in 
an assay is increased to a much higher value than it would be possible to 
achieve at pH 10 in 10-15 minutes. Example 7 demonstrates the increased 
sensitivity of such a method, using a kit in accordance with the 
invention, over the Lowry method. 
It has also been discovered that rapid release of reaction colour at a 
relatively higher alkaline pH, i.e. pH 11-12, results in improved 
reproducibility of protein estimation. Plot A of Example 6 shows a typical 
estimation of protein using an assay kit as described in this invention, 
using a relatively high pH. The points in the plot make a perfect straight 
line, leaving no room for ambiguity. Plot B on the other hand is based on 
the determination at pH 10.5. The points on the plot are scattered and it 
is possible to draw more than one straight line through them. 
When Folin is added to protein treated with alkaline copper, the reagent is 
only reactive for a short time, and it is for this reason that Folin is 
preferably added while vortexing the reaction mixture. It is difficult to 
achieve uniformity with a large number of samples in a batch while adding 
Folin to a vortexing mixture. It has been found that this problem can be 
eliminated by keeping the volume of copper-treated protein small and 
introducing Folin forcibly in a volume larger than the volume of 
copper-treated protein. The forcible addition of Folin creates 
instantaneous mixing of Folin with copper-treated protein which ensures 
uniform mixing of the reagents in a batch. 
Protein assay methods based on the reduction of Folin by copper treated 
protein suffer from interference by a number of commonly used laboratory 
reagents, particularly nonionic and cationic detergents such as 
Triton-X100. This interference can be eliminated by introducing into the 
assay, prior to the addition of Folin, a small amount of an anionic 
detergent such as SDS. This is best achieved by the inclusion of SDS in 
the protein assay kit. Example 8 shows that addition of SDS in an assay 
eliminates interference by Triton-X100. 
It has also been found that it is difficult to maintain SDS in a solution 
of sodium hydroxide having a concentration higher than 0.4N. SDS in as low 
a concentration as 0.5% has a tendency to precipitate on standing in 
sodium hydroxide solution of concentration higher than 0.4N. It is 
therefore preferred that SDS is provided as a separate component in an 
assay kit, and kept separate from the alkaline copper solution, the two 
solutions being mixed prior to use. The SDS solution may be warmed prior 
to use to maintain the SDS in solution. 
SDS can be dissolved either with tartrate or copper sulphate and stored for 
a long time. SDS can also, although this is not preferred, be included in 
an alkaline copper solution and stored for several months (see Example 
11). 
EXAMPLES 
The examples set out below were used to investigate the characteristics of 
a protein assay method, which could be carried out using a kit in 
accordance with the invention. Also investigated were the properties of 
the components of such a kit, of relevance to the preferred 
characteristics of the kit for use in a protein assay. 
Example 11 particularly investigates the stability of an alkaline copper 
solution, for use as one of the components of the kit. 
The materials and methods used in the examples were as follows: 
Reagents 
Copper sulphate pentahydrate, potassium tartrate, sodium tartrate, sodium 
carbonate, sodium hydroxide, sodium dodecyl sulphate (SDS) and bovine 
serum albumin (BSA) were obtained from Sigma Chemical Co. The alkaline 
copper solution was made in two parts, the first part (hereinafter 
referred to as "the alkaline solution") of which contained sodium 
hydroxide, sodium carbonate, sodium or potassium tartrate and SDS. The 
second part was a concentrated solution of copper sulphate. The Folin 
reagent solution was made using a 2N Folin reagent starting solution. 
Reagent Preparation 
Various concentrations of alkaline solution were prepared. They were 0.4N, 
0.8N, 1N and 2N sodium hydroxide solution. Either 4% or 5% sodium 
carbonate and 0.16% sodium tartrate were added to the alkaline solution. 
Similarly, SDS was added to the alkaline solution to a final concentration 
of 0.5% to 2%. The alkaline solution was stored at room temperature in 
polypropylene bottles. A 5% copper sulphate solution was made in distilled 
water and stored at room temperature in a polypropylene bottle. The 
working alkaline copper solution was made by mixing 10 ml of alkaline 
solution with 0.1 ml of 5% copper sulphate solution. 
Folin solutions were made using pure de-ionised water. 2%, 5% and 10% Folin 
solutions were made using a 2N Folin solution and stored at room 
temperature in polypropylene bottles protected from light. Bovine serum 
albumin (BSA) was dissolved in distilled water to a final concentration of 
2 mg/ml and used as standard stock. 
Protein Assay Method 
Protein solutions containing 10 to 200 .mu.g protein in a volume of 0.05 to 
0.2 ml were pipetted into test tubes. The alkaline copper solution was 
added to the test tubes in a volume equal to 1-5 times the volume of 
protein solution in the test tubes, and vortexed. Immediately after 
vortexing, unless otherwise specified, an appropriate volume of Folin 
reagent solution was forcibly introduced into the test tubes. The test 
tubes were incubated for 10 minutes at room temperature and absorbency at 
650-750 nm was measured. The weight of protein was plotted against the 
corresponding absorbence, resulting in a standard calibration curve used 
to determine the amount of protein in unknown samples. 
Micro Protein Assay 
Protein solutions containing 1 to 8 ug protein in a volume of 5 ul were 
pipetted into either micro test tubes or microtiter plates. Alkaline 
copper solution was added into the micro test tubes in a volume equal to 
4-5 times the volume of protein solution, i.e. 20-25 ul. An appropriate 
volume of Folin reagent solution was forcibly introduced into the micro 
test tubes. The micro test tubes or microtiter plates were incubated at 
room temperature for 10 minutes and then absorbences at 650-750 nm were 
read. The weight of protein was plotted against the corresponding 
absorbence, resulting in a standard calibration curve used to determine 
the amount of protein in unknown samples. 
Example 1 
Determination of Short Incubation Period for Protein Assay 
A series of duplicate samples of standard protein solution containing 0.2 
mg protein in a volume of 0.1 ml were treated with 0.5 ml of alkaline 
copper solution containing 0.4N NaOH in 4% sodium carbonate, 0.16% sodium 
tartrate and 0.05% copper sulphate. After mixing, the contents were 
treated with increasing volumes of 2% Folin reagent, introduced forcibly. 
The volume of 2% Folin was increased from 5.2 times the total volume of 
copper-treated protein (i.e. 0.6 ml) to 6.7 times. The optical density was 
read after 10 minutes (Plot B) and after 30 minutes (Plot A) incubation at 
room temperature. The results gave the-plots shown in FIG. 1. As seen from 
the graph, the optical density taken after 30 minutes' incubation (Plot A) 
crosses over the optical density taken after 10 minutes' incubation (Plot 
B). The crossover point is referred to in the graph as "Re" and marked 
with an arrow. The crossover point has a reaction commencing pH of around 
pH 11.70. It is clear from the graph that the reduction of Folin with 
copper-treated protein at pH around pH 11.7 reached its end-point maximum 
in around 10 minutes, and the optical density remained unchanged for the 
next 20 minutes. The reduction of Folin at a pH significantly higher than 
pH 11.70 begins to decline rapidly after 10 minutes and, similarly, the 
reduction of Folin at a pH significantly lower than pH 11.70 continues to 
increase after 10 minutes' incubation. It is clear from these graphs that 
a protein assay method based on the reduction of Folin at a pH around pH 
11.70 can be developed which will reduce the incubation period to around 
10 minutes. At a pH of around 11.70 the optical density stays nearly 
constant for long enough to allow assay of a large number of samples 
without significant drift in determination. 
Example 2 
Determination of Stability of Reaction Colour 
A sample of standard protein solution containing 0.1 mg protein in 0.1 ml 
was mixed with 0.5 ml of alkaline copper solution containing 1N NaOH in 5% 
sodium carbonate, 0.16% sodium tartrate and 0.05% copper sulphate. After 
mixing, 3.55 ml of 5% Folin reagent was forcibly introduced. A 1 ml 
portion was removed for pH determination, and the remainder was used to 
measure the optical density. The optical density was continuously measured 
for one hour. The pH at the commencement of the reaction was measured 
within 1.5 minutes, and was approximately pH 11.70. The optical density 
result gave the plot shown in FIG. 2. It is clear from the graph that the 
reduction of Folin commencing at a pH around pH 11.70 caused rapidly 
increased optical density and reached its end-point maximum in around 10 
minutes, which changed very little for the next 50 minutes. It was 
estimated that, after reaching the end-point maximum in 10 minutes and for 
the next 15-20 minutes, the decline in optical density was negligible 
(around 1%) and in the subsequent 20-25 minutes, it was around 3%-4%. An 
overall drift in the optical density of 3%-4% in one hour is comparable to 
the widely used method by Bradford, M. M. (Anal. Biochem. 1976, 72, 
248-254) and considerably better than the 16% drift in the optical density 
of another popular method by Smith, P. K. (Anal. Biochem., 1985, 150, 
76-85). The results also show that in a 45-minute period, the pH of the 
reaction gradually drops by approximately 0.2 units. 
In a similar experiment to that described above, the protein was treated 
with unbuffered alkaline copper solution containing 0.16% sodium tartrate 
and 0.05% copper sulphate in 1N NaOH. After mixing, 3.7 ml of 5% Folin 
reagent was forcibly introduced and the reaction pH was read within 1.5 
minutes of mixing: this was around pH 11.70. The optical density gave the 
plot shown in FIG. 3. The result shows rapid reduction of Folin, which 
reached a maximum within 10 minutes. The optical density remained 
virtually unchanged for over 30 minutes and, after that, it began to 
decline gradually. In a 60-minute period, the drift in reaction colour was 
around 2%-3%, which is a considerable improvement on the example described 
above. The result also shows that the pH drops by approximately 0.2 units 
in the first 10 minutes, and approximately 0.3 units in 30 minutes, of the 
reaction. 
Example 3.1 
Stability of Optical Density at Various Reaction pHs in Buffered Medium 
A sample of standard protein solution containing 0.1 mg protein in 0.1 ml 
was mixed with 0.5 ml of buffered alkaline copper solution containing 1N 
NaOH in 5% sodium carbonate, 0.16% sodium tartrate and 0.05% copper 
sulphate. The copper-treated protein solution was treated with increasing 
amounts of 5% Folin (3.1 ml to 4.6. ml). The optical density of reaction 
colour was recorded and the pH of the reaction was recorded at intervals. 
The results were tabulated as shown in Table 1. 
TABLE 1 
______________________________________ 
Approximate 
Approximate Approximate 
Length of 
Volume of 
Reaction pH Time to Reach 
Plateau at 
5% Folin 0 45 Maximum Maximum 
(ml) (Minutes) (Minutes) (Minutes) 
______________________________________ 
3.1 12.10-11.95 4-5 4-5 
3.2 12.00-11.88 5-6 6-8 
3.3 11.95-11.75 6-7 8-9 
3.4 11.85-11.62 6-8 10-12 
3.5 11.75-11.58 8-10 20-22 
3.6 11.65-11.40 10-12 25-30 
3.7 11.55-11.30 13-14 40-45 
3.8 11.53-11.30 15-17 &gt;50 
3.9 11.40-11.12 16-18 &gt;60 
4.1 11.23-11.95 18-20 &gt;60 
4.3 11.10- 32-34 &gt;60 
______________________________________ 
It is clear from the Table above that the reduction of Folin with 
copper-treated protein commencing at a pH of between 11.8 and 11.60 
reached its end-point maximum in around 8-10 minutes. After reaching its 
maximum, and for the next 15-20 minutes, the optical density remained 
nearly constant. The deviation in optical density in a 30-minute period 
was negligible at around 1%. The deviation in the subsequent 30 minutes 
was around 3%-4% (not shown in Table 1). The reduction of Folin commencing 
at a pH of between 11.60 and 11.40 reached its maximum in around 15-20 
minutes and stayed nearly constant for 30-40 minutes. In the subsequent 30 
minutes, the decline in optical density was around 3% to 4%. 
It is clear from Table 1 that the higher the alkalinity of the reaction, 
the more rapidly production of reaction colour takes place and, 
conversely, the higher the alkalinity, the shorter is the length of the 
plateau at the maximum optical density. At the reaction pH 11.75, a 
reasonable balance is struck and the reaction takes under 10 minutes to 
reach its maximum colour, while the plateau at the maximum lasts around 
15-20 minutes. The reaction commencing at pH 11.50 would take around 20 
minutes to reach the maximum colour, and the colour at the maximum would 
remain unchanged for over 30 minutes. Lowering the alkalinity 
substantially increases the time it takes to reach the maximum reaction 
colour, although it also increases the stability of the reaction colour. 
It has also been found that the rate of decomposition of the reaction 
colour is considerably reduced by lowering the alkalinity (not shown in 
this table). The reaction colour is stable for over 30 minutes when the pH 
at the plateau is lowered to around pH 11.50 and beyond. It is also clear 
from Table 1 that, in buffered alkaline medium, during the course of 
reaction the pH of the reaction mixture gradually decreases, and in 45 
minutes the pH decreases by approximately 0.2 units. 
Example 3.2 
Stability of Optical Density at Various Reaction pHs in Unbuffered Medium 
A sample of standard protein solution containing 0.1 mg protein-in 0.1 ml 
was mixed with 0.5 ml of unbuffered alkaline solution of copper, 
containing 1N NaOH in 0.16% sodium tartrate and 0.05% copper sulphate. The 
copper-treated protein was treated with increasing amounts of 5% Folin 
(3.4-4.0 ml). The optical density of the reaction colour was recorded, and 
the pH of the reaction was recorded at intervals. The results were 
recorded in Table 2 and FIG. 3. 
TABLE 2 
______________________________________ 
Approx. 
Approx. Reaction 
Approx. Time 
Length of 
pH to Reaction Plateau at 
Volume 5% 
0 15 45 Maximum Maximum 
Folin (ml) 
(Minutes) (Minutes) (Minutes) 
______________________________________ 
3.4 11.95-11.77-11.65 
5-6 10-11 
3.5 11.87-11.70-11.58 
8-9 18-20 
3.6 11.80-11.60-11.50 
9-10 24-26 
3.7 11.75-11.58-11.48 
9-10 26-28 
3.75 11.73-11.53-11.45 
10-11 28-30 
3.8 11.65-11.49-11.33 
16-18 50-55 
4.0 11.55-11.33-11.20 
30-35 &gt;75 
______________________________________ 
It is clear from the results that in unbuffered alkaline medium, the pH of 
the reaction mixture drops more rapidly than in the buffered medium (Table 
1), and a drop of approximately pH 0.2 units takes place in under 10 
minutes. Consequently, when the reaction is commenced at around pH 11.70, 
it rapidly releases the reaction colour and reaches a maximum in around 10 
minutes. The reaction releases acid and as a result, the pH drops to 
around pH 11.50 which happens to be a pH at which the reaction is nearly 
unchanged for well over 30 minutes (Table 1). 
The overall effect is a rapid release of reaction colour at a very high 
alkaline medium (in around 10 minutes) and, as the maximum reaction colour 
is reached, the pH of the reaction drops to a pH at which the 
decomposition of the colour in 60 minutes is insignificant. It has been 
found that in a 60-minute period the drift in optical density is around 
2%. 
Example 3.3 
Rate of Folin Reduction and Stability of Reaction Colour in the absence of 
Tartrate in Unbuffered Alkaline Copper Solution 
The experiments were performed as described in Example 3.2, except that 
tartrate was not added in the alkaline copper solution. The results, shown 
in Table 3, show that in the absence of tartrate the reduction of Folin by 
copper-treated protein proceeds slowly. 
TABLE 3 
______________________________________ 
Approx. 
Approx. Reaction. 
Approx. Time 
Length of 
pH to Reaction 
Plateau at 
Volume 5% 
0 15 45 Maximum Maximum 
Folin (ml) 
(Minutes) (Minutes) (Minutes) 
______________________________________ 
3.4 11.95-11.75-11.61 
10-12 24-25 
3.6 11.78-11.60-11.49 
14-16 28-30 
3.7 11.68-11.48-11.33 
17-18 60-65 
3.8 11.65-11.40-11.30 
25-27 &gt;65 
______________________________________ 
The results in Table 3 show that the reaction commencing at pH 12 took 
approximately 10-12 minutes to reach the maximum as compared to 5 minutes 
in unbuffered and buffered alkaline solution as shown in Tables 1 and 2. 
In addition, in the absence of tartrate, the reaction colour was more 
stable after reaching the maximum value. 
EXAMPLE 3.4 
A Model for Protein Assay at High Alkaline pH 
The results of Example 3.2 were plotted to create a model for protein assay 
at high alkaline pH. FIG. 4 shows the plot. Reaction commencing pH was 
plotted against time. The broken lines show the changes in the reaction pH 
before reaction colour optical density reached a maximum value. The 
semicircle represents the time, corresponding to reaction commencing pH, 
that it took a reaction to reach its maximum value. The solid lines show 
the length of time the optical density of reaction colour remained stable. 
It is clear from the model that the higher the alkalinity of the reaction, 
the more rapidly the release of reaction colour takes place. Conversely, 
the higher the alkalinity, the shorter is the length of stable optical 
density at maximum. When reduction of Folin commences at a pH around 11.7, 
it reaches the maximum reaction colour in approximately 10 minutes. Having 
reached the maximum value, the reaction colour optical density remains 
virtually unchanged for the next 30 minutes. 
Example 4 
Maximising the Release of Reaction Colour 
The experiment consisted of a batch of four determinations, a, b, c and d. 
Duplicate samples of 0.1 ml protein solution containing 0.2-1.2 mg/ml were 
pipetted for each batch. Batches a, b, c and d were treated with 0.1, 0.2, 
0.3 and 0.4 ml alkaline copper solution (containing 0.4N NaOH in 4% sodium 
carbonate, 0.16% sodium tartrate and 0.05% copper sulphate). After mixing 
the contents, 1.16, 1.74, 2.32 and 2.9 ml of 2% Folin reagent was forcibly 
added to a, b, c and d respectively. After an incubation of 10 minutes, 
the optical density was read and the results gave the plots shown in FIG. 
5. 
The next experiment consisted of a batch of three determinations, a, b and 
c. Duplicate samples of 0.1 ml protein solution containing 0.2-1.2 mg/ml 
were pipetted for each batch. The protein solutions were treated with 0.5 
ml alkaline copper solutions, the compositions of which were as follows: 
1. 0.4N NaOH containing 4% sodium carbonate, 0.16% sodium tartrate and 
0.05% copper sulphate; 
2. 1N NaOH containing 5% sodium carbonate, 0.16% sodium tartrate and 0.05% 
copper sulphate; 
3. 2N NaOH containing 5% sodium carbonate, 0.16% sodium tartrate and 0.05% 
copper sulphate. 
Batches a, b and c were treated with alkaline copper solutions 1, 2 and 3 
respectively. After mixing the contents, batches a, b and c were treated 
with 3.5 ml of 2%, 5% and 10% Folin reagent respectively. The optical 
density was recorded and the results gave the plots shown in FIG. 6. It is 
clear from FIGS. 5 and 6 that the release of reaction colour can be 
increased and maximised by increasing either the amount or the 
concentration of alkali and correspondingly increasing the amount of Folin 
solution in the assay. 
Example 5 
Determination of Cu-Protein Complexing Time 
Triplicate protein solutions containing 0.1 mg of protein in 0.1 ml were 
mixed with 0.5 ml of alkaline copper solution containing 1N NaOH in 5% 
sodium carbonate, 0.16% sodium tartrate and 0.05% copper sulphate. The 
contents were mixed immediately and, without delay, 3.5 ml of 5% Folin 
solution was forcibly introduced. The whole procedure took around 15 
seconds to complete. Three more samples were treated identically except 
that Folin solution was added after incubation periods of 1/2, 1 and 10 
minutes. The optical density was read after 10 minutes and the results 
gave the histogram shown in FIG. 7. It is clear from the results that 
copper complexed with protein immediately after the addition and mixing. 
Similar results were obtained when protein was treated with alkaline 
copper solution containing 0.4N NaOH. It is clear that copper complexed 
with protein immediately in alkaline solution containing as little as 0.4N 
NaOH. 
Example 6 
Reproducibility and Accuracy of the Assay 
Reproducibility and accuracy of the assay were examined by performing 
identical determinations. Samples containing 0.025-0.1 mg/ml were mixed 
with 0.5 ml of alkaline copper solution containing 1N NaOH in 5% sodium 
carbonate, 0.16% sodium tartrate and 0.05% copper sulphate. After 
vortexing the mixture, 3.5 ml of 5% Folin solution was forcibly introduced 
into the copper-treated protein solution. The pH of the reaction mixture 
was measured around 11.75. The optical density of the assay reached its 
maximum in around 10 minutes. The optical density was repeatedly read 
after 10 minutes, and the results gave Plot A shown in FIG. 8. An 
identical determination was also performed in which the reaction was 
commenced at pH 10.5, and the results gave Plot B shown in FIG. 7. The 
results show clearly that protein estimation based on the method described 
produces highly reproducible results. In Plot A, the points give a perfect 
straight line and, on the other hand, the estimation based on pH 10.5 of 
the reaction has a larger deviation (Plot B). 
In a further extension of this experiment, the samples of Plot A were 
repeatedly read for an hour, and the results are shown in FIG. 9. The 
results show clearly that in a one-hour measurement, the expected drift in 
the optical density was not detected, and the points were closely packed. 
The inclination of the standard plot remained unchanged. The described 
method is therefore highly reproducible and reliable for estimation of 
protein. 
Example 7 
Sensitivity of the Assay 
The sensitivity of the assay was assessed by comparing it with the results 
produced by the Lowry method. Duplicate samples of protein solution 
containing 0.025-0.1 mg/ml in a volume of 0.1 ml were assayed as described 
in Example 6 and by the Lowry method. The results gave the plots shown in 
FIG. 8. It is clear from the graph that the assay performed according to 
the described method is more sensitive than the Lowry method. 
Example 8 
Elimination of Interference 
Protein assays using Folin reagent are sensitive to interference by a 
number of commonly used laboratory reagents. The small effects due to such 
agents as sucrose, EDTA, Tris and 2-mercaptoethanol can easily be 
eliminated by running a proper buffer control with the assay. The 
interference by nonionic and cationic detergents can be eliminated by 
introducing a small amount of the anionic detergent sodium dodecyl 
sulphate (SDS) (0.5%-2%) into the alkaline copper solution. Duplicate 
samples (0.2-1.2) were assayed as described in Example 6, except that the 
assays were performed with alkaline copper solution containing and lacking 
2% sodium dodecyl sulphate. Protein solutions containing and lacking 1% 
Triton-X100 were used. The results are shown in FIG. 10. Plot B shows the 
control experiment, and Plot D shows the experiment in which the alkaline 
solution contained 2% sodium dodecyl sulphate. The addition of sodium 
dodecyl sulphate slightly increased the colour yield. Plot A shows protein 
containing 1% Triton-X100 when assayed with the reagent lacking sodium 
dodecyl sulphate, and Plot A was distorted because of precipitation due to 
the presence of Triton-X100 in the protein. Plot C shows protein of Plot A 
assayed with the alkaline copper solution containing 2% sodium dodecyl 
sulphate. It is clear from the plots that the addition of sodium dodecyl 
sulphate countered the influence of detergent Triton-X100 and restored 
Plot A. Similar results have been obtained with other nonionic and 
cationic detergents. 
Example 9 
Micro Assay System 
Micro protein assay was performed in either microtiter plates or micro test 
tubes in a total assay volume of 0.2-0.25 ml. Protein solution containing 
0.2-1 .mu.g protein in a volume of 5 .mu.l was used. The protein samples 
were first treated with 25 .mu.l of alkaline copper solution containing 1N 
NaOH in 5% sodium carbonate, 0.15% sodium tartrate and 0.05% copper 
sulphate followed by 174 .mu.l of 5% Folin solution. The optical density 
was read after 10 minutes. FIG. 11 shows the results obtained for a micro 
assay system. It is clear from the results that the assay is capable of 
estimating protein at concentrations as low as 0.2 micrograms in a sample. 
Example 10 
Linearity of the Assay 
This experiment consisted of a batch of five determinations, a, b, c, d and 
e. Batch a contained 0.2-1.2 mg/ml protein in a total volume of 0.1 ml. 
Batches b, c, d and e contained 2, 3, 4 and 5 times the amount of protein 
present in Batch a in a total volume of 0.1 ml. The assays were performed 
as described in Example 6, and FIG. 12 shows the results. It is clear from 
the results that the assay is linear for up to 0.6-3.6 mg/ml protein per 
assay. Protein in excess of 0.8-4.8 mg/ml begins to lose linearity. 
However, it has been found that the linearity of the assay can be restored 
by increasing the amount of alkaline copper in the assay and 
correspondingly increasing the amount of Folin (results not shown). 
Example 11 
Stability of Alkaline Copper Solution 
Unbuffered alkaline solution containing 1N NaOH and 0.16% tartrate was 
mixed with copper solution in order to prepare alkaline copper solutions. 
0.5% SDS was added to some of the alkaline copper solutions. The 
solutions-alkaline copper solution lacking in SDS and alkaline copper 
solution containing 0.5% SDS--were routinely examined for signs of 
deterioration. It was found that alkaline copper solutions did not develop 
excessive precipitate and were good for use for months. However, the 
alkaline copper solution containing SDS did develop SDS precipitate, 
although the solution could still be used after shaking it gently, which 
created a homogeneous suspension, or after warming, which dissolved the 
SDS precipitate and gave a clear solution. It was also found (see Table 4) 
that the presence of a high concentration of tartrate further improved the 
stability of an alkaline copper solution. In the absence of tartrate, the 
copper precipitated out of the alkaline solution as a black precipitate. 
TABLE 4 
______________________________________ 
The effect of concentration of sodium/potassium tartrate 
on the stability of an alkaline copper solution. The 
alkaline copper solution contained 0.05% copper in 1N 
NaOH solution. The reagents were stored at room 
temperature over 6 months. 
Concentration 
Na/K tartrate 
Concentration 
in alkaline copper 
of tartrate 
solution relative to Remarks of 
(%) copper & Results 
______________________________________ 
0 0.sup. Black precipitate developed 
within hours 
0.16 .times.3.2 Black precipitate developed 
within weeks 
0.26 .times.5.2 Black precipitate in 
some samples 
0.66 .times.13.2 All samples clear. No 
precipitation observed. 
______________________________________