Bioluminescence method for the determination of pesticides

A method for the rapid and sensitive determination of organophosphate and carbamate pesticides is disclosed. The method employs an insect brain preparation having a mixture of receptors or enzymes with sites that interact with the organophosphate and carbamate pesticides. The pesticides alter or reduce brain activities of the insect brain preparation which are inversely correlated with pesticide concentration in a test sample. The activity is measured by employing substrates which upon exposure to the insect brain preparation are structurally altered and a light emission reaction is observed. The substrates used are 6-substituted D-Luciferin esters wherein the D-Luciferin ester in the presence of the insect brain preparation is inhibited during hydrolysis while in the presence of the pesticide to be determined. Further the D-Luciferin ester in the presence of the insect brain preparation reacts to cleave the substituted ester group at the 6-position to produce D-Luciferin for further reaction with ATP and luciferase to produce bioluminescence. The bioluminescence can then be measured and compared with a control in order to determine the concentration of a pesticide in the test sample.

DESCRIPTION 
The parent application concerns a radio assay test method employing 
C.sup.14 radiolabeled materials which includes adding insect brain tissue, 
such as bee or housefly brain tissue homogenate, to a test sample 
containing an organophosphate or carbamate pesticide which interacts with 
the brain homogenate. The admixture is incubated and then a radiolabeled 
substrate, for example, a C.sup.14 substrate, is added to interact with 
the remaining sites in the incubated insect brain homogenate and the 
mixture incubated. The resulting incubated test sample, brain homogenate 
and radiolabeled substrate is then separated to provide a liquid fraction 
sample to which is added a scintillation material. The radioactivity of 
the separated fraction is then determined by counts per minute and 
compared with a standard or control to determine the concentration of the 
pesticide in the test sample. 
BACKGROUND OF THE INVENTION 
Different kinds of organisms (arthropods, avians, mammals) are sensitive to 
pesticides. Pesticides are generally classified as herbicides, fungicides 
and insecticides. Pesticides interact with their nervous and enzymatic 
systems. Such toxicants may bind to binders (ion channels) located on the 
nerve cells, or to enzymes located around them and elsewhere. Pesticides 
also interact with various protective mechanisms, such as degrading 
enzymes and non-specific binders. 
The brain and the nerve system of insects in general were the target for 
insecticides since their early development (1940s) for pest control in 
agriculture and human health. Pesticides that target the insect brain as 
the site of action usually display rapid action and require low dosage for 
good control. 
Since the early 1970s, attempts have been made to use in vitro brain 
preparation to study the mode of action of pesticides. Only limited 
studies were reported on using this method for monitoring pesticides. Lack 
of stability and sensitivity as compared with the traditional gas 
chromatography (GC) or mass spectrometry made this early attempt useless. 
The use of color reaction requires high enzume and substrate concentration 
to monitor brain preparation activity and the results were insensitive 
assays. The enzymes used were taken through laborious purifications, and 
it was found that only a limited spectrum of pesticides could be detected. 
Apparently, the source and the purified preparation were of limited 
sensitivity and for only a few pesticides which made it unsuitable as a 
monitoring and screening assay. 
In recent years, bioluminescence or chemiluminescence has been used to 
detect extremely small quantities of material. Assays using these signals 
have been developed to measure substrates, such as ATP or NADH, and 
enzymes, such as alkaline phosphatase. Advantages to using bioluminescence 
as an assay system is the sensitivity of the reaction and the speed in 
which that reaction can be measured. For example, the bioluminescence 
reaction with the enzyme luciferase catalyzes the reaction between 
luciferin and ATP to produce light in milliseconds. 
It is desirable to test for the concentration of pesticides in various 
materials, such as soil and water for health and safety purposes. In 
particular, it is desirable to provide an effective test kit and method to 
determine the concentration level of organophosphate and carbamate 
pesticides at low levels, such as below 50 ppb and even as low as 5 ppb. 
Specific organophosphate insecticides may be tested employing antibodies, 
but these have limited use as broad spectrum screening methods. Herbicides 
may be tested on a specific basis by chromogenic enzyme-based test 
methods, but such tests do not provide accurate results at low 
concentration levels and are susceptible to color interpretation. 
Therefore, a new, accurate, effective test kit and method for the 
determination of pesticides, such as organophosphate and carbamate 
pesticides, are desirable. 
SUMMARY OF THE INVENTION 
The invention relates to a test kit and method for the determination of 
pesticides. In particular, the invention concerns a chemiluminescence or 
bioluminescence test method and test kit for the determination of 
organophosphate and carbamate-type pesticides at levels below about 50 
ppb. 
A test kit and method has been developed for the rapid, generally 10 to 15 
minutes, and sensitive, less than 50 ppb or lower, method for the multiple 
detection of organophosphate and carbamate pesticides in water, soil, food 
(e.g. meat, fish, fruits and vegetables) and other materials. The test 
method employs an insect brain preparation having a mixture of receptors 
or enzymes with sites that interact with the pesticides and particularly 
that react or interact with organophosphate and carbamate pesticides. The 
pesticides alter or cause a significant reduction in the brain activities 
of the insect brain preparation which are inversely correlated with the 
amount of the pesticide present in the test sample. The activity is 
measured by employing tracer analogs or substrates which upon exposure to 
the insect brain preparation are altered chemically and/or physically and 
the changes monitored by a light emission reaction. The activities can be 
chemical as in an enzymatic reaction or physical as in receptor binding. 
For example, in one embodiment in an enzymatic reaction, the hydrolysis of 
a luciferin derivative to luciferin by a brain preparation has been found 
to be extremely sensitive to organophosphates and carbamates. The 
luciferin liberated by the reaction is oxidized by the enzyme luciferase 
and adenosine triphosphate (ATP), an energy donor, and emits light 
measured as bioluminescence or chemiluminescence. The bioluminescence or 
chemiluminescence emitted is measured and monitored at a low level and 
high speed employing a luminometer or scintillation counter. The assay is 
carried out in three simple steps of a short incubation period, e.g. 2 to 
10 minutes, of the test sample and the brain preparation; addition of a 
substrate tracer or pesticide analog tracer, such as a luciferin 
derivative with additional incubation, e.g. 2 to 5 minutes; and the 
measurement directly or after a separation step to obtain liquid fraction 
for light emission by activation with the enzyme (luciferin-luciferase). 
The general assay preparation is illustrated in Table 1. 
Therefore, a test method has been found for the determination of pesticides 
which are sensitive to insect brain preparation and particularly 
organophosphate and carbamate pesticides. The test method comprises 
incubating a mixture of a test sample and an insect brain preparation; 
adding to the incubated mixture a D luciferin derivative, such as, but not 
limited to: D luciferin acetate, whose hydrolysis is inhibited in the 
presence of the pesticide; incubating the test sample, brain preparation 
and D luciferin derivative mixture to liberate luciferin; admixing a 
portion of the incubated D luciferin derivative-containing admixture with 
ATP and luciferase as a reaction mixture to provide an oxidized luciferin 
(oxyluciferin) and emitted luminescence; measuring the emitted 
luminescence, for example, with a luminometer; and determining the 
concentration of the pesticide in the test sample by comparison of the 
emitted, measured luminescence of a standard or of a control sample. 
The test kit for the determination of pesticides comprises in combination 
an insect brain preparation which is sensitive to the pesticide, e.g. bee 
brain homogenate, a D luciferin derivative which is inhibited in 
hydrolysis in the presence of the pesticide, such as D luciferin acetate; 
the enzyme luciferase and ATP to form a reaction mixture when added 
together to the incubated test sample, brain preparation and D luciferin 
derivative. The test kit may include those standard articles of laboratory 
equipment and chemicals, like buffers, needed to carry out and measure the 
results to include, but not be limited to: incubation dishes or plates and 
an incubation water bath, etc.; buffers; a luminometer to measure emitted 
luminescence; a standard control chart or graph of luminescence vs. 
pesticide concentration for comparison and determination of the pesticide 
concentration; and separating equipment, such a chromatographic column or 
ultrafiltration membrane to obtain a liquid fraction of the incubated 
mixture. 
A wide variety of insect brain tissue material may be used in the test 
method of the invention, such as the crude, stabilized brain tissue, 
particularly the homogenate of arthropods, for example, but not limited 
to: bees; beetles; aphids; mosquitoes; silkworms; mites; blow flies; and 
houseflies (Musca domestica) alone or in combination. 
Brain preparations from various sources have differences in specificity for 
pesticides. Therefore, it is important to obtain one or more brain 
preparations which will react with a broad spectrum of pesticides for 
monitoring purposes. Insects as the target for pesticides are one of the 
best sources for brain preparations. Specificity and sensitivity can vary 
from one insect to another. Bees were chosen as a preferred choice for 
their known sensitivity to a variety of insecticides. It was found that 
bee brain preparation was approximately 2 to 4 logs more sensitive in the 
bioluminescence assay system than any previously reported assay system. 
One D luciferin derivative suitable for use in the pesticide test method 
comprises 6-acetyl D luciferin or luciferin acetate. Suitable luciferin 
derivatives are those 6-substituted D luciferin compounds which in the 
presence of insect brain preparation react to cleave the substituted ester 
group at the 6-position of the D luciferin derivative to provide D 
luciferin for further reaction with ATP and luciferase to produce 
luminescence which can be measured. The luciferin derivative 6-acetyl D 
luciferin is prepared by reacting D luciferin with excess acetyl imidazole 
in a solvent, e.g. water-solvent solution which acetylates D luciferin 
under mild conditions with about 100% efficiency to provide the 6-acetyl D 
luciferin. 
It has been found that both organophosphate and carbamate pesticides 
inhibit the hydrolysis of D acetyl luciferin by insect brain preparations. 
There are significant advantages in the use of a D luciferin derivative, 
like D luciferin acetate, as a substrate in the test method. There is an 
unusual, unexpected, high specificity of the bee or silkworm brain 
preparations toward D luciferin acetate and only small quantities of the D 
luciferin acetate are required for the assay. For example, the 
concentration of D luciferin acetate in the incubation mixture may be as 
low as 1.times.10.sup.-11 moles or less and the assay sensitivity for 
luciferin is 1 to 5.times.10.sup.-13 moles. The assay time is about 10 
minute in total. While D luciferin acetate has been found to be a 
preferred D luciferin derivative for use in the test method, other D 
luciferin derivatives with similar reactions may be employed. Those 
luciferin derivatives disclosed in the publication of the Journal of 
Clinical Chemistry and Chemical Biochemistry entitled "Synthesis and 
Characterization of Luciferin Derivatives for Use in 
Bioluminescence-Enhanced Enzyme Immunoassay New Ultrasensitive Detection 
Systems for Enzyme Immunoassay", Miska et al, J. Clin Chem Clin Biochem 
25(1) 1987, pp. 23-30, are not suitable for use in the test method and 
include specifically D-luciferin methyl ester, 
D-luciferyl-L-phenylalanine, D luciferyl-L-Na-arginine, 
D-luciferin-O-sulphate and D-luciferin-O-phosphate. 
To test the ability of the assay system to monitor for the presence of 
pesticides in food, six apples from different sources were tested. Of the 
six apples, four apples were positive and two apples were negative. The 
four positive apples were obtained from local orchards, while the negative 
apples were purchased from local supermarkets. Local water was also tested 
and was found marginal positive. Table 3 gives detection levels expected 
in water for 15 different organophosphate and carbamate pesticides. 
Test Procedure Used for Organophosphate and Carbamate Pesticides 
1. Sample is preincubated with a predetermined quantity (dilution 1:200) of 
brain preparation for 5 minutes. 
2. Luciferin acetate (200 pmoles) is added to the incubation mixture for 
additional 5 minutes. 
3. A portion of this invention mixture (100 .mu.l) is withdrawn and added 
to the luciferase and ATP reaction mixture (1 ml). 
4. Bioluminescence reading for 2-5 seconds is monitored. 
5. The reading of the pesticide sample is compared to the control sample to 
quantitate the percent inhibition of the sample (see Tables 3, 4, 5 and 6) 
.

The invention will be described for the purposes of illustration only in 
connection with certain embodiments; however, it is recognized that those 
persons skilled in the art may make various improvements, additions, 
changes and modifications to the illustrated embodiments all falling 
within the spirit and scope of the invention. 
DESCRIPTION OF THE EMBODIMENTS 
A. Synthesis of luciferin acetate (6-acetyl D luciferin) 
Dissolve 2 mg sodium D-luciferin (purified from firefly) into 2 ml water, 
or dissolve 2 mg synthetic D-luciferin into 2 ml methanol as stock 3.3 mM 
luciferin solutions in an amber vial. Take 25 .mu.l of either stock 
solution and add it to 1 ml distilled water in an amber microcentrifuge 
tube to get an 82.5 .mu.M solution of luciferin. Dissolve 60 mg of 
N-acetylimidazole into 1.0 ml acetone to get a 545 mM solution of 
N-acetylimidazole. Add 30 .mu.l of 545 mM solution of N-acetylimidazole to 
the 82.5 .mu.M aqueous solution of luciferin. Mix several times and 
monitor the decrease of bioluminescence as the reaction continues to 
completion. The reaction is complete when bioluminescence can no longer be 
observed when using the above reaction mixture as a source for luciferin. 
Keep solution on ice. The concentration of N-acetylimidazole in this 
reaction mixture is 16.35 mM or approximately 200 times the concentration 
of luciferin. 
Notes: D-Luciferin from firefly was purchased from Sigma. Synthetic 
D-luciferin was purchased from Boehringer Mannheim. Also, a D-luciferin is 
available from Bio-Orbit. Luciferin stock solution from Sigma was 
dissolved in water while D-luciferin stock solution from boehringer or 
Bio-Orbit was dissolved in methanol. N-acetylimidazole was purchased from 
Sigma. The reaction described above was performed in 1 ml and 1 
concentration range, but it could be scaled up using a larger volume size. 
Other derivatives of luciferin, such as luciferin phosphate, luciferin 
sulfate, luciferin arginine have been described in the literature, but to 
out knowledge, luciferin acetate has not been described, nor to our 
knowledge has it been used in a coupled reaction with brain extract to 
monitor pesticides. 
B. Preparation of Insect Brain Extract 
Insects are stored from at -20.degree. C. or below. Insect heads are 
collected by dissection with a scalpel and placed in a 50 ml beaker 
containing approximately 15 ml ice cold 0.07M phosphate buffer, pH 7.0, 
containing 1 mM EDTA (ethylenediaminetetra acetic acid) and 1 .mu.M 
phenylthiourea. The insect heads are gently homogenized at low speed in a 
Tekmar Tissumizer. Aliquots of this crude extract are transferred to 
microcentrifuge tubes and centrifuged. The supernatant is retained and 
applied to a sephadex G-25 column equilibrated with 0.07M phosphate 
buffer, pH 7.0, containing 1 mM EDTA and 1 .mu.M phenylthiourea. Fractions 
are collected every 2 minutes and monitored for esterase activity using 
luciferin acetate as a substrate and measuring bioluminescence. Fractions 
with esterase activity are pooled and used as the insect head extract for 
use in the pesticide assay. 
Notes: Brain extracts have been prepared primarily from honey bees, but 
also has been extracted from silkworm and blowflies. Protein concentration 
of the bee head extract is between 2 to 5 mg/ml. For the assay, 5 to 10 
.mu.l is used per assay. 
C. Bioluminescence Reaction 
1. Reaction buffer for bioluminescence: Weigh out 4.48 g tricine, 0.6 g 
magnesium sulfate, 0.146 g EDTA, 100 mg bovine serum albumin, and 77 mg 
dithiothreitol into 600 ml water. Adjust pH to 7.8 with 10% sodium 
hydroxide and add distilled water to 1 liter. 
2. Luciferase preparation: For luciferase (1 mg) from firefly purchased 
from Boehringer Mannheim dissolve in 1 ml 0.5M Tris-acetate buffer, pH 
7.5, and let stand 30 minutes. Portion out 40 .mu.l of this solution into 
glass test tubes and freeze at -20.degree. C. Redissolve frozen stock with 
1 ml bioluminescence buffer. For luciferase purchased from Bio-Orbit take 
3 mg and dissolve in 1 ml bioluminescence buffer. Each luciferase solution 
is kept on ice. For each bioluminescent assay, a 30 .mu.l aliquot from 
either luciferase solution described above is used. For the Boehringer 
luciferase, approximately 1 .mu.g enzyme is used for each assay. 
3. ATP Preparation: ATP (adenosine 5'-triphosphate) is preweighed from 
Sigma and contains 1 mg ATP and 40 mg magnesium sulfate. To this vial add 
10 ml water and 100 .mu.l 1.0M HCl and vortex. ATP solution is kept on 
ice. The stock solution is 1.8 mM and for each bioluminescent assay, 30 
.mu.l is used for a final concentration of 54 .mu.M. 
4. Luciferin Acetate: Take 100 .mu.l 82.5 .mu.M solution of luciferin 
acetate prepared as described above in section A and 900 .mu.l distilled 
water and add to amber microcentrifuge tube. This solution is 8.25 .mu.M. 
For pesticide assays, a 25 .mu.l portion of the 8.25 .mu.M solution is 
added to 2 ml 0.07M phosphate buffer, pH 7.0, to give a 0.1 .mu.M 
concentration of luciferin acetate. A 100 .mu.l portion of this solution 
is withdrawn and incubated with 1 ml of the bioluminescent buffer. Final 
concentration is approximately 10 nM luciferin acetate or 10 pmoles 
luciferin acetate. 
5. Bioluminescence Measurement: A 1 ml portion of the bioluminescent buffer 
is added to a 13.times.100 mm test tube. A 100 .mu.l portion of the 
luciferin acetate solution (0.1 .mu.M) is withdrawn and added. Then 30 
.mu.l aliquots of the luciferase and ATP solutions are added to the test 
tube. The test tube is vortexed and the bioluminescence measured after 2 
to 5 seconds using a luminometer. The bioluminescence reading with 
luciferin acetate will be negative. If luciferin is measured at 10 pmoles 
then the reading in the luminometer will be approximately 100,000. If the 
concentration of any of the reagents described above is increased, then 
the light reading will be increased. Although the present concentrations 
give acceptable values, the concentration of the reagents may be altered 
to give optimal performance. The above liquid reagents may also be 
immobilized in individual tablet form to provide stability and consistency 
to the reagents, such as in compressed tablet form with inert cellulosic 
fillers. 
D. Assay of Pesticides Using Bioluminescence 
1. To 13.times.100 mm test tubes add 2 ml 0.07M phosphate buffer, pH 7.0. 
Add 25 .mu.l positive pesticide control (organophosphate and/or carbamate 
pesticide), 25 .mu.l water to negative control tube and 25 .mu.l of test 
sample to another tube. If test sample contains solvent other than water, 
then use this solvent as the negative control tube. 
Note: Although 25 .mu.l is indicated above, the volume size of sample could 
be less or greater as long as brain activity is not affected by the 
solvent. 
2. Add insert brain extract (5 or 10 .mu.l depending on activity) to the 2 
ml incubation mixture in step 1, vortex and incubate at 35.degree. C. in 
temperature block for 10 minutes. Use timer with alarm to monitor time. 
3. At 10 minutes, add 25 .mu.l 8.25 .mu.M luciferin acetate solution to 2 
ml incubation mixture in step 2. Reset timer to zero and restart timer. 
4. At 5 minutes, take 100 .mu.l portion from each sample. Use separate 
pipette tip for each sample and add the 100 .mu.l portion to separate 
13.times.100 mm test tubes containing 1 ml of bioluminescence buffer. 
Note: In this reaction, the sample is withdrawn after 5 minutes. Since the 
reaction is kinetic, the concentration of luciferin formed in the reaction 
will increase with time. Therefore, the time an aliquot can be withdrawn 
can vary as long as it is in the linear section of the curve, and aliquots 
from samples are withdrawn at the same time. 
5. Add 30 .mu.l ATP solution followed by 30 .mu.l luciferase solution to 
one of the assay tubes in step 4. Vortex and measure bioluminescence for 2 
to 5 seconds. Record value and proceed to the next tubes as above. 
6. Zero control bioluminescence reaction will be uninhibited by pesticide 
and will have a high bioluminescence reading. In the positive samples the 
esterolitic activity of the bee extract will be inhibited and less 
luciferin will form giving a low bioluminescent reading. Divide sample 
reading or positive control by zero control and multiply to 100 to get 
percent inhibition. 
Note: The source for some of the chemicals used has been set forth; 
however, other suppliers can supply these chemicals and therefore, the 
procedure is not reliant on any one source for a particular reagent except 
for the preparation of luciferin acetate. 
Tables 5-11 represent test data showing the increase in bioluminescence as 
a function respectively of time, luciferase, ATP, luciferin acetate and 
bee head brain preparation in the test assay. 
TABLE 1 
______________________________________ 
Schematic of Pesticide Assay 
______________________________________ 
1. BE.sup.a) + pesticide.sup.b) (brain extract) 
##STR1## BE - pesticide + BE 
2. D luciferin derivative.sup.c) 
##STR2## D luciferin + luciferin derivative 
3. ATP + luciferin (adenosine triphosphate) 
##STR3## oxyluciferin + pyrophosphate + AMP 
(adenosine monophosphate) + luminescence (to 
be measured).sup.d) 
______________________________________ 
Legend 
.sup.a) for example, insect bee brain homogenate 
.sup.b) organophosphate and carbamate pesticides 
.sup.c) for example, D luciferin acetate 
.sup.d) luminometer 
TABLE 2 
______________________________________ 
PREATION OF D LUCIFERIN ESTER 
______________________________________ 
##STR4## 
##STR5## 
##STR6## 
##STR7## 
______________________________________ 
LEGEND 
"R" IS AN ALKYL GROUP e.g. C.sub.1 -C.sub.6 OR PHENYL GROUP 
TABLE 3 
______________________________________ 
Charm Pesticide Assay for Organophosphate and Carbamate 
Inhibition of Bee Brain Activity for Ac-luciferin 
I.sub.50.sup.a) 
Pesticide (ppb) 
______________________________________ 
Carbamates 
Methomyl 4 
Propoxur 10 
Carbofuran 8 
Bendocarb 12 
Organophosphates 
Mevinphos 2 
Ethion 2 
Chlorpyrifos 1 
Phorate 2 
Malathion 6 
Oxydemeton-methyl 1 
Disulfoton 5 
Methyl parathion 1 
DDVP 0.004 
Naled 0.05 
Diazinon 15 
______________________________________ 
.sup.a) I.sub.50 is the concentration of inhibitor which gives a 50% 
decrease in enzymatic activity as measured by bioluminescence. 
TABLE 4 
______________________________________ 
Inhibition of Silkworm Brain Activities by 
Various Organophosphate Pesticides at 25 PPB 
% 
Pesticide Inhibition 
______________________________________ 
Phorate 50.5 
Naled 98.0 
Methyl parathion 0 
Ethion 77.0 
Oxydemeton-methyl 
0 
Diazinon 0 
DDVP 100.0 
Disulfoton 25 
Mevinphos 50 
______________________________________ 
TABLE 5 
______________________________________ 
Inhibition of Bee Brain Extract by Organophosphates 
As a Function of Pesticide Concentration 
% % % 
ppb activity 
ppb activity 
ppb activity 
______________________________________ 
Methyl Parathion 
Phorate Chlorpyrifos 
______________________________________ 
0 100 0 100 0 100 
0.625 62 1.25 70 0.625 68 
1.25 40 2.5 29 1.25 34 
2.5 23 3.125 23 2.50 24 
5.0 15 6.25 8 3.125 13 
12.5 3 6.25 10 
______________________________________ 
Ethion Diazinon Oxydemeton-methyl 
______________________________________ 
0 100 0 100 0 100 
0.3125 84 25 40 0.3125 90 
0.625 71 50 45 0.625 43 
1.25 65 100 28 1.25 47 
2.5 37 2.5 37 
25.0 21 
______________________________________ 
Disulfoton Mevinphos Malathion 
______________________________________ 
0 100 0 100 0 100 
3.125 66 0.3125 64 6.25 53 
6.25 47 0.625 52 12.5 50 
12.5 35 1.25 48 25.0 27 
25.0 19 2.5 24 50.0 27 
3.125 12 
6.25 9 
______________________________________ 
DDVP Naled 
______________________________________ 
0 100 0 100 
0.0013 74 0.031 50 
0.0023 49 0.625 29 
0.0028 60 0.125 30 
0.0057 52 0.250 25 
0.011 25 
______________________________________ 
TABLE 6 
______________________________________ 
Inhibition of Bee Brain Extract by Carbamates 
As a Function of Pesticide Concentration 
Bendiocarb 
Methomyl Carbofuran Propoxur 
% % % % 
ppb activity 
ppb activity 
ppb activity 
ppb activity 
______________________________________ 
0 100 0 100 0 100 0 100 
12.5 50 3.125 45 6.25 59 3.1 62 
25 26 6.25 16 12.5 41 12.5 43 
50 18 12.5 20 25 32 25.0 44 
100 11 25.0 12 50 22 62.5 29 
125.0 27 
______________________________________ 
TABLE 7 
______________________________________ 
Increase In Bioluminescence as a Function of Time 
Using Pesticide Assay in Control Reaction 
Time (min) Light reading 
______________________________________ 
0 0 
0.75 390 
1.75 1230 
2.75 3030 
3.75 3510 
5.0 5550 
7.0 9360 
9.0 11460 
11.0 13200 
14.0 20640 
16.0 27300 
______________________________________ 
TABLE 8 
______________________________________ 
Increase in Bioluminescence as a Function of Luciferase 
Using the Pesticide Assay in Control Reaction 
Luciferase (g) light reading 
______________________________________ 
0 0 
0.17 390 
0.34 1200 
0.5 1470 
0.67 4830 
0.83 7740 
1.0 8580 
1.17 17280 
1.34 20700 
1.51 31868 
1.68 40140 
______________________________________ 
TABLE 9 
______________________________________ 
Increase in Bioluminescence as a Function of ATP 
Using the Pesticide Assay in Control Reaction 
ATP (.mu.M) Light Reading 
______________________________________ 
0 0 
0.9 390 
1.8 1289 
3.6 4110 
5.4 8220 
9.0 20760 
10.8 24060 
______________________________________ 
TABLE 10 
______________________________________ 
Increase in Bioluminescence as a Function of Luciferin Acetate 
Concentration Using the Pesticide Assay in Control Reaction 
Luciferin Light 
Acetate (.mu.M) 
Reading (at 3 min) 
______________________________________ 
0 0 
0.2 8520 
0.4 15120 
0.8 61680 
1.6 180240 
3.2 467280 
6.4 724080 
______________________________________ 
TABLE 11 
______________________________________ 
Increase in Bioluminescence as a Function of Bee Brain Extract 
Concentration Using Pesticide Assay in Control Reaction 
Bee Light 
Extract (.mu.l) 
Reading 
______________________________________ 
0 0 
10 1920 
20 6484 
40 22260 
80 28880 
______________________________________