Computer-driven amino acid indexer for peptide synthesis

An automated, computer-driven amino acid indexer for peptide synthesis uses programmed computer, a circuit board controller, and a combination of microtiter sample well trays, light emitting diodes to illuminate each sample well, and circuitry to control the illumination of the diodes. The apparatus simplifies technical difficulties present in large-scale laboratory syntheses of peptides by substantially reducing the time required for dispensing amino acids into sample trays and reducing the occurrence of error in the process to negligible levels in typical syntheses. A programmed, automated technique for synthesizing peptides is also provided.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention is in the field of solid-phase peptide synthesis. More 
specifically, this invention relates to a novel computer-driven amino acid 
indexer for rapidly synthesizing peptide samples on a solid support with 
very high amino acid sequence accuracy. 
2. Description of the Related Art 
Solid-phase methods of peptide synthesis have been developed in which 
proteins of defined amino acid sequence are prepared by the step-wise 
addition of amino acids in the defined order to a growing peptide chain on 
a solvent-resistant matrix. De novo design and synthesis of peptides has 
become an indispensable technique in scientific research for a variety of 
important reasons. 
The design and synthesis of novel and naturally occurring peptides makes it 
possible to study protein topologies and conformations as well as the 
kinetics of protein folding. Additionally, many biologically active 
substances, such as hormones and signal molecules, are peptides. Further 
peptide synthesis is useful in identifying the receptor sites on cell 
surfaces where hormones and signal molecules are active. There is keen 
interest in this field because synthetic peptides may be useful as 
pharmaceutical agents with a variety of applications, such as vaccines 
effective against bacterial and viral infections. And synthetic peptides 
may also be used to identify specific antibody binding sites, known as 
epitopes, on antigens, disease-inducing foreign organisms or viruses. As a 
corollary to this last field of endeavor, it is also possible to use 
synthetic peptides as antigens or as fragments which are effective epitope 
mimics to stimulate the production of particular antibodies by the host 
organism (Scientific American Feb. 1983:48-56; Nature 306:9 [1983]). 
The overall immune systems of higher animals comprise very complex 
interactions of non-specific defensive scavengers (phagocytes), specific 
defenders (i.e., antibodies), mediators, and modulators. The capability of 
higher animals to fight a given disease is greatly dependent on the 
ability of the host's antibodies to recognize antigens and bind them 
tightly and specifically. This binding activity precipitates a sequence of 
events that leads to the neutralization and elimination of the organism or 
virus responsible for the disease. Diseases which are subject to 
surveillance by the host immune system in this general manner include, by 
way of example, influenza, tetanus, polio, smallpox, and many cancers. 
Vaccines and therapeutic compounds are being sought for some cancers, 
acquired immunodeficiency syndrome (AIDS), hepatitis B, herpes and other 
diseases. The effects of certain chemical warfare agents are also being 
studied to determine whether such agents are subject to resistance by 
antibodies according to the general scheme described herein. 
A particularly challenging endeavor in the field of immunology is to 
characterize mechanism and binding specificity of a host's immune response 
to a particular peptidic antigen. Each antigen binding site, or epitope, 
is specific to a particular antibody. Solid-phase peptide synthesis 
techniques are especially useful in this endeavor, in that a synthetic 
peptide having the same amino acid sequence as an epitope, or one so 
similar that it effectively mimics the epitope, will bind an antibody in 
the same tight and specific manner as will a natural antigen possessing 
the epitope. Peptide fragments which copy of mimic an epitope can trigger 
the same immune response as a peptidic antigen containing the epitopic 
center as well as the genetic material required to replicate itself. Thus, 
immunization by using peptide fragments substantially reduces the risk of 
infection or deleterious side effects. By making up sample peptides and 
mapping their antibody binding characteristics through analytical 
techniques such as enzyme-linked immunosorbent assay (ELISA) or 
radioimmuno assay (RIA), it is possible to characterize the antibody 
reactivity of a large number of peptides and identify those which are 
active as epitopic centers. 
The antibody reactivity mapping technique may be used in conjunction with a 
technique recently introduced by Hendrik Mario Geysen for synthesizing and 
testing large numbers of peptides (U.S. Pat. No. 4,833,092, May 23, 1989, 
Method for Determining Mimotopes, and U.S. Pat. No. 4,708,871, Nov. 24, 
1987, Antigenically Active Amino Acid Sequences). In this technique, 
peptides are synthesized incrementally backwards according to processes 
known in the art of peptide synthesis chemistry. An activated derivative 
of the correct individual amino acid is pipetted onto each growing peptide 
chain, each peptide being synthesized separately on a plastic pin. An 
8.times.12 or similar matrix of pins may be used in the syntheses. After 
hundreds of peptides are individually prepared, each on its own respective 
pin, the pins are assayed by ELISA for antibody reactivity. The peptides 
made according to this method are typically eight amino acid residues in 
length and have an overlapping pattern along the entire protein sequence. 
(The testing of an antigen containing 100 residues in a defined sequence 
according to the overlapping pattern would require synthesizing 
eight-residue test samples, beginning with one corresponding to residues 
1-8 of the antigen, the next corresponding to residues 2-9 of the antigen, 
then 3-10, through the last peptide which would correspond to residues 
93-100 of the antigen). 
Since the pioneering work of R. Bruce Merrifield in developing a 
solid-phase peptide synthetic method using solvent-resistant polystyrene 
beads, Science 232:341-347 [1986], techniques of peptide synthesis have 
remained essentially labor-intensive and time-consuming. This means that 
skilled technicians must spend long periods of time in performing 
mechanical tasks, such as pipetting amino acid derivatives into a matrix 
of solvent-resistant wells for incremental growth of peptide chains in 
desired sequences. Matching the properly ordered amino acid with the 
proper well in the matrix has remained essentially a manual task. With 
current techniques, even an experienced technician can suffer from mental 
fatigue and confusion during synthesis of multiple peptide samples, one 
hundred to one thousand, for example, while trying to keep the different 
wells identified and the prescribed amino acid sequences straight. 
Notwithstanding the experience and skill required of the technicians so 
employed and the time that such mechanistic processes inherently require, 
error rates have typically exceeded the rates that are normally considered 
acceptable in a biochemistry laboratory. It is apparent that committing 
trained and skilled technicians to laborious and tedious occupations is 
inefficient and undesirable. 
Given the keen interest in peptide synthesis and analysis shown by 
government, industry, and academic institutions, it is highly apparent 
that simplified means for peptide synthesis which overcome the technical 
problems associated with current techniques are needed. Simplification can 
reduce technical errors in the sequencing of amino acids during synthesis, 
save time, help to reduce human error evident in tasks such as the set up 
of synthetic apparatus, and free technical personnel for other tasks. In 
this invention, an amino acid indexer for peptide synthesis is provided 
which simplifies the various technical difficulties associated with 
peptide synthesis and renders the process more efficient by saving time, 
reducing errors, and lowering the level of skill absolutely required to 
perform the mechanical functions of peptide synthesis. 
SUMMARY OF THE INVENTION 
Synthesis of peptides for epitope mapping analysis, vaccine preparation, 
and other purposes is a vitally important scientific technique which has 
been characterized by tedious manual labor and relatively high error 
rates. By automating the indexing of amino acid sequencing and selection, 
peptide syntheses may be accomplished rapidly with undetectable error. 
The present invention is directed primarily to an apparatus for the 
computer-driven indexing of amino acids for large scale laboratory 
synthesis of peptides. The apparatus provides computer programmed amino 
acid selection and sequence indexing with a combination of 
solvent-resistant sample trays, light means, main circuit control means 
for receiving indexing signals and translating them into control signals 
for the light means, and latch circuit board control means for governing 
the light means in accordance with the signals received from the main 
circuit control means. In one aspect of the invention, there are ten 
sample plate arrays each comprising an 8.times.12 matrix of wells, ten 
latch circuit control means and ten 8.times.12 arrays of light means, 
providing one light means such as a light emitting diode for each sample 
well. In this aspect, there are sample trays and automated control means 
for the preparation of 960 different polypeptides. 
In another of its aspects, the invention is directed to an automated, 
computer-driven method for preparing peptides comprised of different amino 
acid sequences.

DETAILED DESCRIPTION OF THE INVENTION 
This invention pertains to a computer-driven amino acid indexer designed 
primarily for the synthesis of peptides used in epitope mapping of protein 
antigens. The indexer of this invention is designed to allow for more 
rapid synthesis of peptides than by prior art techniques, which are 
manually intensive procedures, while reducing error to a negligible rate. 
Standard ELISA and RIA microtiter plates may be used with the indexer of 
this invention. Computer software can be designed to make the indexer 
applicable to a wide range of biological and biochemical investigations, 
including: lymphocyte proliferation, cell surface receptor binding, 
cytotoxicity, virus plaques, enzyme kinetics, and other procedures often 
performed in clinical chemistry and microbiology laboratories. Almost any 
large experiment that requires pseudo-random filling of microtiter plates 
lends itself to the amino acid indexer of this invention with appropriate 
software applications. Thus, it is an object of this invention to provide 
an apparatus for increasing the accuracy and efficiency of peptide 
synthesis for epitope mapping. It is a further object of the invention to 
provide an apparatus and a method to aid researchers in the quick, 
efficient, low cost development of peptides leading to faster vaccine 
development. Details of various embodiments of the invention will be set 
forth in detail below. 
The amino acid indexer of this invention is designed to simplify various 
technical problems of matching the twenty different amino acids to 
potentially hundreds of microtiter plate wells for each day's work of 
peptide syntheses while incidentally reducing human error in apparatus 
set-up in some instances. It is well known that error rates for satellite 
and digital data transmission average about 1 bit per billion bits of data 
transmitted (1:10.sup.9 bits), and it is reasonable to forecast that the 
electronic error propagated by a 3-foot length of transmission cable, 
which would be typical of an apparatus of this invention, would be 
substantially lower. A complete eight-day peptide synthesis run like the 
one described below in the Example would require the transmission of 
153,000 bits of data; at the generally recognized error rate of 1:10.sup.9 
bits, more than 6,500 replications of that eight-day synthesis could be 
made with the erroneous transmission of only one data bit. This error rate 
is obviously negligible in a statistical sense, but in a qualitative sense 
it is negligible also, in that the error is unlikely to distort epitope 
mapping by eliminating a possible mimotope. 
To achieve the objectives, an apparatus for use with computer software and 
hardware, such as IBM PC/XT/AT compatible for IBM PS-2 series computers, 
has been developed. The indexer comprises a plurality of plates, each with 
a multiplicity of separate wells. A light source such as a light emitting 
diode (LED) is mounted in each well, thus allowing each well to be 
illuminated individually when the light source is so instructed by a 
computer. The configuration of the amino acid indexer and its interaction 
with machine driven sequencing programs will now be described in further 
detail with reference to the figures accompanying this disclosure. 
FIGS. 1 and 2 show, respectively, top and side views of light source arrays 
for microtiter plate well arrays (not shown) and related components of 
this invention. A sample plate with microtiter wells arrayed in this 
instance in a 8.times.12 well matrix is situated immediately atop an LED 
board 5 with an LED element 10 corresponding to each plate well in the 
array of the sample plate. The LED board 5 has a support structure 8 and 
the LED elements may optionally have collars 6 to collimate light beams 
under the microtiter plate wells. Pin connectors 9 connect the LED board 5 
to a latch circuit board 2. The general configuration of chips 4 is shown 
on the latch circuit board 2 in both figures. The plate arrays are 
composed of these main elements: a sample plate, an LED board 5 (which 
includes an LED element 10 for each well in the array) and a latch circuit 
board 2 to drive the LEDs. Preferably, these elements are stationarily 
situated upon a base structure. Where multiple arrays are used, as is 
desired, the respective latch boards are connected to each other and to 
the main control board (not shown in FIGS. 1 and 2) with bus cables (also 
not shown). 
Circuit boards are shown in schematic diagrams in FIGS. 3 and 4. The main 
control board of the invention is shown in FIG. 3. The control board 
comprises several chips which are identified here with reference numerals 
corresponding to those in the figure. Data is received by a receiver chip 
12 which converts the voltages to transistor-transistor logic (TTL) levels 
(for example, +5 vdc and 0 vdc). The signal is fed to a universal 
asynchronous receiver/transmitter (UART) chip 14. A baud rate generator 
chip 16 provides the correct clock frequency to the UART chip 14 to 
synchronize the rate of transmission of data bits from the UART with that 
being received from the computer. The UART output goes to an octal driver 
chip 18 which drives the data through a bus cable (not shown) to each 
latch board 2. A row counter chip 20 is incremented every time the UART 
chip 14 receives a character. Row count information is expressed in four 
data lines that are bussed to each of the latch boards 2. A 3-input AND 
gate 22 is used to reset the row counter chip 20 to zero. A one-shot chip 
24 is pulsed when the counter chip 20 is reset, incrementing a one-of-ten 
counter chip 26. This chip is used to track which microtiter well plate is 
currently active. The ten data lines from the one-of-ten counter chip 26 
pass through inverters 28 and 30 and are bussed to each latch board. The 
latch boards, depicted in FIG. 4, comprise a 4 to 16 line data distributor 
3, twelve octal latches 4 and inverters 7. A one-shot chip 32 initializes 
all counters to zero when the apparatus is first turned on, and inverters 
34 provide the correct polarity to chips 14, 20, and 26. 
The latch boards 2 are chained together and connected to the main control 
board by a bus cable. The computer sends data via signals to the main 
control board using RS-232 asynchronous communication protocol. Where a 
8.times.12 matrix is used, such as the sample plate matrix described 
above, a standard 8 bit American Standard Code for Information Interchange 
(ASCII) code may be used. In this code, a single 8-bit character 
represents the light pattern of each row of each 8.times.12 LED matrix 
(each bit corresponds to a single well). If a bit has a high TTL level (+5 
vdc, for example), the corresponding LED will be off; if a bit has a low 
TTL level (0 vdc, ground, for example), the corresponding LED will be 
illuminated. If, as described here, ten plates each having an 8.times.12 
matrix are used, then the main control board transmits to the indexer 130 
ASCII characters for each amino acid, or one character for each of twelve 
rows for each of the ten plates, plus one null character per plate to 
reset the counter. 
The bus cable is comprised of twenty-six conductors. Eight lines carry the 
character data which actually illuminates the LEDs in the proper sequence 
in accordance with the amino acid profile in the computer. Ten lines carry 
the active plate information (where ten different plates are employed) 
while four lines carry the plate row data and 4 lines are used to provide 
the high voltage and ground signals. Each latch board has access to all 
the bus information simultaneously. The individual latch boards 2 are 
addressed (from 1 to 10 where a ten board apparatus is in use) by a 
shorting block 11. The addresses correspond to the ten active plate lines 
from the main control board. When the active plate line corresponding to a 
latch board address is high (+5 vdc), the 4 to 16 line data distributor 3 
on the latch board is activated. The data distributor 3 decodes the four 
plate row data lines and after being inverted (inverters 7) activate the 
corresponding octal latch which latches the character data from the bus 
and illuminates the appropriate LEDs. As each character is passed down the 
data bus, it is passed onto one of the twelve octal latches 4 on one of 
the latch boards 2. The interrelationships of the programmed computer, the 
main control circuit board, and the latch circuit boards of the invention 
are depicted in the flow chart set forth in FIG. 5. 
Apparatuses for peptide synthesis comprising a computer, plate well arrays, 
and illumination means for wells may be used in conjunction with a variety 
of main circuit board control means and electronic latching means 
differing from the circuits depicted in FIGS. 3 and 4 and described 
herein. In a preferred embodiment, the main circuit control board 
comprises a TTL receiver chip for receiving amino acid sequencing and 
selection data impulse signals from a computer, a universal asynchronous 
receiver/transmitter chip, a baud rate generator chip, an octal driver 
chip, a row counter chip, a 3-input AND gate, a one-shot chip, a 
one-of-ten counter chip, and ten inverters. The latch boards of the 
preferred embodiment comprise a 4 to 16 line data distributor chip, twelve 
octal latches, and two inverters. The latch boards are connected to each 
other and to the main control board in the preferred embodiment by a bus 
cable comprised of conductors for each of the plates to carry plate 
activity data, for each row of the standard matrix to carry LED 
illumination data, for plate row selection and for high or ground voltage 
data. Where ten plates, each comprising an 8.times.12 plate well matrix, 
are employed, the bus cable would have twenty-six conductors: ten for 
plate activity, eight high/low voltage data for LED illumination, four for 
plate row selection, two for power and two for ground. In the preferred 
embodiment, each LED board and its respective latch control board are 
connected by two 50-pin connectors 9. Ten plates, each having an 
8.times.12 plate well matrix, capable of carrying the synthesis of 960 
different peptide chains, are employed in this apparatus. 
A particular application of the apparatus and method of this invention in 
synthesizing peptides according to the 8-catamer system introduced by H. 
M. Geysen, cited above, is presented in the following Example. 
EXAMPLE 
Software was designed specifically for synthesizing peptides according to 
the Geysen system using an apparatus of the invention and following the 
method of the invention. The software program reads a protein sequence, in 
this case the protein sequence shown in FIG. 6, and creates separate 
pattern files. Since peptides containing eight residues each were being 
prepared in this Example, eight separate pattern files were created (one 
day was devoted to placing an amino acid residue in each well according to 
the sequence instructed by the computer, so eight days in all were 
required for the syntheses of this Example). Pattern files contain 
information to instruct the indexer apparatus as to which amino acid 
should be placed in which plate well on each of eight succeeding days of 
the synthesis exercise. The logic of the software designed for mimotope 
peptide synthesis as performed in this Example is depicted in the flow 
chart set forth in FIG. 7. The amino acid index apparatus used in this 
example employed ten solvent-resistant microtiter plates, each comprising 
an 8.times.12 matrix of wells. Solvent-resistant empty plates were laid 
atop LED indicator plates, each having a 8.times.12 matrix of LEDs to 
correspond to the well matrix arrangement, allowing LEDs to illuminate 
their corresponding wells individually. 
The software designed for this exercise provided five main menu choices of 
function: (1) Create Peptide Sequence; (2) Implement Sequence; (3) Print 
Report; (4) Test Hardware; and (5) ELISA Analysis. The apparatus was 
assembled as described, viz., with 8.times.12 solvent-resistant plate 
arrays placed atop 8.times.12 LED arrays, connected to an LED latch 
control board. Ten plate arrays containing 96 wells each were set up. The 
plates were connected to the main control circuit with a twenty-six 
conductor bus cable. An IBM PC AT was used. Solutions containing each of 
the twenty amino acids were prepared. 
The synthesis sequence was begun by selecting menu choice (1), Create 
Peptide Sequence. Once the peptide sequence was created, the program 
created a comprehensive report detailing each peptide string that was to 
be synthesized, sized, the amounts of the respective amino acids that were 
to be used each day to complete the syntheses, and the eight pattern files 
that would be used to light the LEDs on the amino acid indexer in the 
patterns required for synthesis of 960 unique peptide strings. The report 
may be printed at the option of the researcher (menu choice (3)). 
Synthesis of peptide strings was begun with the selection of menu choice 
(2), Implement Sequence. The program prompted the researcher to the 
correct pattern file for the appropriate day of the synthesis exercise. 
When the pattern file for the appropriate day was selected, a list of the 
twenty amino acids was displayed to the screen. The researcher can select 
amino acids in any order for placement in the solvent-resistant microtiter 
plate wells. When the Insert key was depressed, the software sent 
appropriate signals to the amino acid indexer to illuminate the wells into 
which reagent containing the amino acid selected was to be deposited. When 
the researcher had dispensed reagent containing the acid into each 
illuminated well, the Insert key was again depressed and another 
illumination pattern corresponding to a different amino acid was 
displayed. Reagent containing this acid was dispensed into each 
illuminated well. This procedure was repeated for each of the twenty amino 
acids until all 960 wells had been filled with its one amino acid. To 
effect peptide chain growth on the solid phase support, a 
solvent-resistant support suitable for ELISA analysis, such as a 
polystyrene bead or a polyethylene pin, was placed in each well after the 
researcher had dispensed therein the first amino acid residue. The 
Sequence Implementation procedure was repeated each day for eight days to 
complete the peptide syntheses. The finished peptide remains covalently 
bound to the support. It may then be analyzed on the solid phase or 
chemically cleaved and tested in solution. 
Referring now to FIG. 6, the sequencing worked in the following manner: 
Looking at the code symbols for the first twenty amino acids of the test 
protein we see the following sequence: 
MKKIMLIASA MSALSLPFSA 
For the first eight peptide strings containing eight amino acids each 
(discounting controls comprising smaller fragments that were used in the 
exercise), the software signalled the indexer to prepare peptide strings 
in the following sequences pursuant to the overlap pattern introduced by 
Geysen: 
1. MKKIMLIA 
2. KKIMLIAS 
3. KIMLIASA 
4. IMLIASAM 
5. MLIASAMS 
6. LIASAMSA 
7. IASAMSAL 
8. ASAMSALS 
Since the peptides were synthesized in reverse order, amino acids A,S,A, 
M,S,A,L,S were delivered to wells 1 through 8, respectively, of the first 
plate on the first day of synthesis. This is confirmed by reading the one 
letter code for the last amino acid of each peptide fragment vertically 
down the list of eight given above, from top to bottom. During the first 
day's synthesis, when the researcher requested the LED pattern for Alanine 
(A) to be displayed, the bit pattern 01011011 was sent to the first row 
and LEDs in positions 1,3, and 6 were lit, indicating that a solution 
containing Alanine was to be placed in wells 1,3 and 6 of the first plate 
(a 0 in the bit pattern indicates a lit LED). Continuing with this 
illustration, on the second day when the researcher requested the LED 
pattern for Arginine (R), the bit pattern 11111111 was sent to the first 
row; when the LED pattern for Alanine (A) was requested, the bit pattern 
10101101 was sent to row 1 of plate 1, indicating that a solution 
containing the amino acid Alanine was to be placed in wells 2,4 and 7. It 
is important to note that this brief illustration covers the first eight 
peptides while the indexer is designed always to receive (in a 960 
fragment synthesis using ten 8.times.12 well arrays) 120 8-bit patterns 
for each amino acid (8 rows.times.12 columns.times.10 plates). To complete 
the illustration, 119 appropriate bit patterns would have been sent to the 
indexer for each acid after the first row bit patterns were sent, as 
described above. The software used in the syntheses of this Example 
created the appropriate number of day files (8 in this instance), each 
consisting of 2400 bit patterns (120 patterns.times.20 amino acids). To 
simplify the process, each 8-bit pattern was represented by its ASCII 
character equivalent, thus allowing the use of existing DOS operating 
system software to send the pattern files to the indexer. 
The overlapping pattern continued in the manner following this pattern 
through the synthesis of 960 peptide strings beginning with the first 
eight amino acids of the protein sequence of FIG. 6. The syntheses were 
completed in one-sixth the time that purely manual syntheses of the same 
number of peptide strings would have taken, at a negligible error rate. 
Having been given the teachings of this invention, variations and 
ramifications will occur to those skilled in this art. Thus, even though 
highly specific circuit configurations for the main control board and the 
latch boards have been described in a preferred embodiment, other 
arrangements are obviously feasible. Further, whereas an apparatus with 
ten plates, each having 96 wells arranged in 8.times.12 matrices, has been 
illustrated, other matrices and differing numbers of plates may be 
employed. Additionally, although syntheses of 8-residue peptides following 
Geysen were illustrated, the apparatus and method can be used in other 
biochemical techniques, including syntheses of peptide strings having more 
or less then eight residues each. Different kinds of software can be 
custom designed to meet the particular needs of specific applications of 
the apparatus and method of this invention. These and other modifications 
occurring to those skilled in the art are deemed to be within the scope of 
this invention.