WO1992021959A1

WO1992021959A1 - Method and apparatus for electrochemical determination of biological substances

Info

Publication number: WO1992021959A1
Application number: PCT/US1991/002448
Authority: WO
Inventors: Ricardo J. Moro
Original assignee: Diagnostic Concepts International, Inc.
Priority date: 1991-06-06
Filing date: 1991-06-20
Publication date: 1992-12-10
Also published as: AU8301191A

Abstract

There is described a new and improved method and apparatus for performing an enzyme-linked immuno sorbent assay of a biological substance in a fluid substrate wherein an enzyme conjugate is used to react with the biological substance to cause the release of ions into the substrate. The change in the resistivity of the substrate due to the release of ions is measured and an analog signal is generated in response thereto. The analog signal is converted into a digital signal which is quantized and output in a human-readable form for indicating the quantity of the biological substance present in the substrate.

Description

METHOD AND APPARATUS FOR ELECTROCHEMICAL DETERMINATION OF BIOLOGICAL SUBSTANCES

Field of the Invention

"he present invention relates to a method and apparatus for the detection of organic substances, and more particularly relates to an improved ELISA reader for detecting trace amounts of biological substances in a sample.

Background of the Invention

ELISA (Enzyme Linked Immuno Sorbent Assay) test procedures have been developed for the detection of organic substances in blood and other bodily and biological fluids. The ELISA technique is based upon three primary principles: first, the ability of proteins to bind to plastic; the high affinity and specificity of antigen/antibody reactions; and the ability of enzymes to modify a substrate. ELISA assay procedures are well known and will not, therefore, be described in great detail herein. Briefly, however, a plastic plate having typically ninety-six individual plastic wells is the receptacle for the fluid sample. Each of the ninety-six wells is coated with an antibody and an antigen specific to the antibody is bound thereto to hold the antigen in place. A second antibody specific to the same antigen is added and bonded thereto. The second antibody has been covalently conjugated prior to its introduction with an enzyme which will react with a substrate to cause a color change therein given the right environmental conditions. The degree of color change in the substrate is proportional to the amount of the biological substance such as an antigen sought to be detected. By passing filtered light through the substrate and measuring its degree of absorption by the substrate using an ELISA reader, one can calculate the concentration of the biological substance therein as a function of the amount of absorbed light which is assigned a numerical value or index. The greater the color intensity of the substrate, the greater absorption of light.

The use of ELISA techniques has increased greatly having regard to the procedure's relative simplicity, speed, reliability, sensitivity and as a means of avoiding the use of radioactive assays.

The disadvantages of ELISA readers that measure light absorption are several. The readers themselves are delicate and sensitive and require constant adjustment to maintain optimal sensitivity. The linear response of the readers falls within a very narrow range so that each test requires large numbers of dilutions so that at least one or two of such dilutions falls within the linear range of the instrument. A relatively intense light source is required making it more difficult to obtain a portable unit having regard to the inherent power requirements of the system, and of course the plastic lens at the bottom of each well must pass the light without distortion, necessitating the use of relatively expensive optical grades of plastic which further adds to the costs. The readers themselves are relatively expensive (prices range from $6,000. for relatively simple models, up to $28,000. for units with computerized data management and analytical capabilities) and some existing readers perform only one light measurement at a time, making the reading of a plate with ninety-six wells a relatively time-consuming process.

Summary of the Invention

It is an object of the present invention to obviate and mitigate the disadvantages of existing ELISA readers and provide an improved reader of equal or greater sensitivity which is faster to use, requires less maintenance and energy, possesses greater linearity, is less costly to build and is more amenable to the construction of a portable unit.

In accordance with these objects, it has been found advantageous to produce an analyzer reader that measures, not light absorption but, rather, the conductivity or, conversely, the resistivity or the impedance of the substrate following enzymatic reaction. As will therefore be appreciated, the present invention contemplates the use of enzyme conjugates which promote the formation of ions in the substrate, rather than color reactions. One such conjugate uses urease which catalyzes the transformation of urea into ammonium carbonate. The ammonium is present in the substrate in the form of ions which change the conductivity/resistivity of the substrate. This change can be measured and is directly proportional to the amount of biological material present in the substrate that is to be measured. Quantifying the change will produce a measurement of the biological substance useful to the tester. Because the power requirements for the proposed system are very low, the development of a portable unit is greatly facilitated. According to the present invention, then, there is provided in a method of performing an enzyme-linked immuno sorbent assay of a biological substance in a substrate, the improvement comprising utilizing an enzyme conjugate to react with the biological substance to cause the release of ions into the substrate, measuring the change in resistivity of the substrate due to the presence of the ions, generating an analog signal in response to the measurement of resistivity, converting the analog signal into a digital signal, and quantizing the digital signal and outputting the same in human-readable form for indicating the quantity of the biological substance present in the substrate.

According to a further aspect of the present invention, there is also provided an apparatus for performing an enzyme-linked immuno sorbent assay of a biological substance in a fluid substrate, comprising means for measuring the resistivity of the substrate and producing an analog signal representative thereof, converter means for converting the analog signal into a digital signal, and processing means to process the digital signal to produce numerical data indicative of the quantity of the biological substance in the substrate.

Brief Description of the Drawings

Preferred embodiments of the invention will now be described in greater detail, and will be better understood when read in conjunction with the following drawings, as follows:

Figure 1 is a bottom plan view of a two-pin electrical probe forming part of the proposed ELISA reader.

Figure 2 is a top plan view of the probe of Figure 1. Figure 3 is a block circuit diagram of a computer- based ELISA reader in accordance with one aspect of the present invention.

Figure 4 is a schematic diagram of the ELISA reader card circuit for computer installation. Figure 5 is a block circuit diagram for a portable/hand-held ELISA reader in accordance with another aspect of the present invention.

Figure 6 is a schematic diagram of the portable reader of Figure 5. Figure 7 is a schematic diagram of a first part of a modified embodiment of an ELISA reader card circuit for use with a computer. Figure 8 is a schematic diagram of the second part of a modified circuit for use with a computer.

Figure 9 is a partly schematic, bottom plan view of an electrode head with a matrix of electrodes thereon.

Detailed Description of the Invention

The basic ELISA test procedure described above for colormetric measurement is followed in the present instance with the exception that the enzyme conjugate used to induce color reactions (usually peroxidase antibody or alkaline phosphatase) is replaced with, for example, a urease conjugate for transforming urea into ammonium carbonate. The presence of ammonium ions in the substrate raises its Ph, which can be detected using well known techniques, but this provides only a visual confirmation of the presence of the ions without a quantitative measurement of concentrations. The other change in the substrate is, as aforesaid, to its resistivity/conductivity which can be measured in a quantitative sense. Both urea and pure water are highly resistive, such that a change in conductivity is directly related to the concentration of ammonium ions in solution which, in turn, is a function of the amount of biological material present to be measured. The use of other conjugates for detecting different substances, but which nevertheless transform a non-ionized substrate into one which is of course contemplated, and the foregoing and subsequent descriptions relating to the use of urease are intended to be exemplary in nature only.

With reference now to Figures 1 and 2, there is shown an electrical probe 10 having ninety-six pairs of conductive pins or electrodes 11 corresponding to the ninety-six wells on the plastic plate (not shown) . The pairs of pins 11 are arranged into twelve columns 1-12 and eight rows A-H, with the pins in each column being connected in parallel and thence to one of electrical contacts 14, as shown in Figure 2, and the pins of each row being similarly connected together and thence to an electrical contact 15, as shown in Figure 1. By switching between a pair of contacts consisting of one of contacts 14 and one of contacts 15, each of the ninety-six pin pairs 11 can be individually selected.

In one embodiment of the invention, as contemplated by the applicant, the switching between pin pairs and data acquisition is computer-implemented. The computer-based reader card is illustrated schematically with reference to Figures 3 and 4. An IBM XT (Trademark of IBM Corporation) or compatible computer may be used for this purpose and the card may be plugged into one of the expansion slots therein for direct input of the data into the computer.

Probe 10 is placed in contact with the plastic well plate so that each of pin pairs 11 can measure the resistivity of the solution in its respective well, this resistivity being a function, as aforesaid, of the presence of ammonium carbonate ions released into solution as a result of the enzymatic reaction.

The computer itself is programmed to sample and analyze the data whereby each of the pairs of electrodes 11 is sequentially connected to an analog/digital converter through an electronic inverting switch which prevents polarization of the electrodes. On command, address decoder 20, comprising two 74138 and one 7402 integrated circuits, is activated. In writing mode with the IOW terminal active, timer 22 is activated to enable multiplexers 24 and 25, both of which are latched to the data on the input bus 26 by the IOW instruction. The bus data determines the pair of pins 11 on probe 10 to be read with multiplexer 24 selecting one of columns 1 to 12, and multiplexer 25 selecting one of designated rows A to H. Analog switch 32 connected to both of the multiplexers flips the polarity of pins 11 at a rate of approximately 700 Hz to prevent potentially deleterious sustained polarization of the electrodes. Switch 32 is itself pulsed to flip the polarity of the electrodes by means of a square wave generator 34 which produces the 700 Hz signal required for this purpose.

The analog resistivity measurement taken by the pairs of pins determines the time period that the output of timer 22 is high. That is, the duration of the timer's output signal is determined by the resistivity of the solution in the measured well. The output is connected to a three- state line driver 38 (a 74244 IC) which outputs a digital signal to bus 26. When the timer is on, the data on input bus 26 will be 00000001, which value is read in accordance with the computer program which activates the driver through an IOR command to allow the computer to read the number on bus 26. If the number on the bus is 1, the program remains in a loop which subtracts one unit to a preset variable. Accordingly, when the number on the bus equals 0, the program exits and the variable's value will be directly proportional to the resistance of the substrate. The presence of a relatively large number of ammonium ions will, of course, decrease the resistance of the substrate and will cause a small decrease in the variable's initial value. Conversely, a weak reaction will cause more loops to decrease the variable's initial value to produce a smaller final value. The values so produced are outputted in some useful human-readable form or stored in memory for subsequent analysis.

It is noted that measuring electrolyte conductance using a timer as configured in the present embodiment, as compared to many conventional methods such as resistor bridges, provides a very large dynamic range without the need to switch components (resistors) . A very large resistance (low conductance) in the solution being tested keeps the timer 22 ON for a prolonged period of time, thus yielding a high count output. On the other hand, a very small resistance (a very high conductance) in the solution still provides a measurable period of time during which the timer 22 is ON. The dynamic range is thus only dependent on the speed and the maximal number of counts an associated counting device can achieve and current electronic technology allows several orders of magnitude more speed and counting capacity than necessary for electrolyte conductances measurements in accordance with the present invention.

Another difference between using the timer 22 and conventional ways of reading conductance (bridges) is the voltage applied to the electrodes. In most conventional systems, the voltage applied to the solution is a few millivolts (typically 1-50 mV) . This makes signal amplification mandatory before any processing can be done (direct measurement with an instrument, digitalization, etc.). Should the resistance in the solution be high, then the amplification circuit can be represented as a high impedance input amplifier. A high input impedance makes any amplifier very prone to noise. However, if a square pulse alternating current (very rich in harmonics) is used to reverse the electrodes (to prevent polarization) , the spikes caused by the switching ON and OFF of the pulses may become a source of noise, depending on their amplitude and the amplifiers gain. With the timer 22, the voltage applied to the electrodes is the same as the power supply feeding the timer. This is normally 5 Volts. No amplification is required, thus the problem of noise interference is eliminated along with the complexity added by the amplifying circuit.

The low power requirements of the present method and apparatus facilitate the development of a hand-held unit for portable operation. The circuitry for such an apparatus will now be described with reference to Figures 5 and 6.

A suitable two-pin electrical probe (not shown) is immersed in the well to be measured, the probe being shaped to fit the well as required. The measurement is initiated by closing a switch 54 referred to as a reading switch, which triggers logic gate unit 40 to deliver a LOW output signal to a timer 41 and HIGH signal to a predetermined number such as 9999. Timer 41 is actuated by the LOW signal from gates 40 to deliver a HIGH signal of its own to downcounters 42 which begins to count down from 9999. The same HIGH signal from timer 41 simultaneously disables display drivers 43 to burn LCD display 44 off during the reading period. When the reading has been completed, the timer's LOW signal deactivates the downcounters and reactivates drivers 43, and hence LCD 44, to display the values present at the outputs of downcounters 42.

The time interval that drivers 43 are disabled by timer 41 is related to the resistance of the sample being measured in the well, in which the electrodes of the probe are immersed, and the value of capacitor Cl. When drivers 43 are disabled, downcounters 42 count down the pulses from an 8 KHz clock generator 46. If the reaction in the well is strong and a relatively large number of ions are released into solution, the resistivity of the substrate will be low and the signal from timer 41 will disable drivers 43 and enable downcounters 42 for a relatively short period of time. The number of downcounted pulses from clock generator 46 will be relatively few and, consequently, the number output on LCD 44 will be large. Conversely, if relatively few ions are released into solution whereby its resistivity will be relatively high, timer 41 will disable drivers 43 and enable downcounters 42 for a proportionately longer period of time, with the result that a greater number of pulses from clock generator 46 will be downcounted to produce a lower numerical reading on LCD 44. The response of the portable reader is linear so that the reading on LCD 44 will be directly proportional to the strength of the enzymatic reaction-producing ions which, of course, is a function of the relative concentration of the particular biological substance being tested for.

Once again, it is important that no sustained polarization of the electrodes immersed in the substrate occurs. The voltage across the electrodes is therefore inverted at a rate of approximately 1 KHz by means of electronic switch 45 driven directly by clock generator 46 or indirectly by the clock generator through an invertor 47 to alternatively invert the connection of the pins to timer 41 and the voltage + V.

Clock generator 46 provides a further 60 Hz to drive the backplane of LCD 44.

As contemplated by the applicant, the portable reader can be constructed to read the ninety-six wells of a standard plate one at a time or in multiples of up to and including all ninety-six wells.

Illustrated in Figures 7 and 8 is a modified embodiment of a circuit 100 for use in the invention in the alternative to the circuit illustrated in Figure 3 and partly in Figure 4. The circuit 100 is used in preference to the circuit of the earlier embodiment where there is likely to be a large variance in resistivity of analytes in various wells that could influence the resistance results from adjacent wells.

The principal difference between the circuit 100 and that shown in Figure 4 is that the present circuit 100 uses six 16 channel analog multiplexers 102 through 107, as compared to the pair of 8 and 16 channel multiplexers 25 and 24 respectively that are shown in Figure 4. The multiplexers 102 through 107 are shown in Figure 8 and provide for ninety-six total channels corresponding with the ninety-six wells of a standard plate within which ninety-six analytes are placed for testing. The circuit 100 is divided into first portion 106 which is mounted on a card for insertion into a conventional expansion slot in a personal computer and is illustrated in Figure 7. Likewise, a second portion 107 of the circuit 100 is mounted on a card and illustrated in Figure 8 along with portions from a piggyback card.

With reference to Figure 7, a bus 109 is mounted on the left hand side of the card and connected to a pair of decoders 110 and 111 operate together as a hexadecimal address decoder. A decoder 113 decodes the directional flow of information from or to the computer. The direction decoder 113 functions in conjunction with the address decoders 110 and 111 to determine the direction of information that is passed to it from the computer. The output of this decoding process is used to direct a bidirectional bus driver 115 and a latch 117. The circuit lines which carry data from the bidirectional bus driver 115 and latch 117, as well as the decoding lines (write $14B, write $18B, read $14B and read $18B) and the input power lines, are connected to the remainder of the apparatus by a connector 120 through a suitable twenty-five line connector cable.

With reference to Figure 8, on the left hand side is shown a connector 130 for connecting with the connector 120 shown in Figure 7. The highest bit from the connector 120 (D7) is used by the circuit 100 to detect the status of the output of the address decoder 111. The rest of the circuit lines from the connector 130 are grounded through resistors in order to produce a zero value. The connector 130 is joined to the multiplexers 102 through 107 by circuit lines. In particular, the latched lines (DLO through DL7) are used to select which multiplexer 102 through 107 respectively is enabled. The enabled line is decoded by a decoder 132 in cooperation with the output of a timer 134 such that none of the multiplexers 102 through 107 are left on in between readings. Shown in Figure 9 is an electrode head 150 having ninety-six electrodes 151 and ninety-six companion electrodes 152 arranged in respective pairs and in a matrix adapted to be placed in a prearranged set of ninety-six solution-filled wells for testing. A respective one of each of the multiplexers 102 through 107 connects to one of the ninety-six electrodes 151 through a circuit line identified as Probe 2 in Figure 8 and headers 155 and 156 shown in Figure 9. Each of the electrodes 151 along with a respective companion electrode 152 is associated with sampling of a particular one of the ninety-six wells. The companion electrodes 152 are tied together by a circuit line identified as Probe 1 in Figure 8. In this manner, upon selection of a particular electrode 151 by one of the multiplexers 102 through 107, the respective electrode 151 and its companion electrode 152 which are necessary to read the resistance in a particular likewise-numbered well are connected to the circuit lines Probe 1 and Probe 2. As with the previous embodiment, the resistance generated through the selected well is utilized as the R component in the RC network that determines the length of the pulse of the timer. In order to prevent electrode polarization, a quad bilateral switch 137 in Figure 8 switches the polarity of the electrodes approximately five hundred to two thousand times per second. The switching frequency is determined by timer 138 operating in an astable mode and flip-flop 139 acting to divide the frequency rate in half while assuring a perfectly square wave. Headers 140 and 141 connect the multiplexers 102 through 107 to the respective electrodes. This is accomplished by piggybacking the circuit board containing the remainder of the circuit shown in Figure 8 with a second circuit board including headers 140 and 141 underneath.

The circuit 100 is controlled by a personal computer as has been described for the previous embodiment. Initially, a number between zero and ninety-five and representing the range of ninety-six possible and numbered testing wells is written to address $18B. This number is then latched by latch 117 which selects, in conjunction with decoder 132, which pair of electrodes 151 and 152 is associated with the respective selected well. Simultaneously, timer 134 is triggered which enables decoder 132. Data line D7, which is connected to the output of timer 134, goes HIGH. This information is passed by driver 115 to the bus 109 when a read $18B operation is done. A loop in the computer program polls address $18B and a unit is added to an energy variable every turn of the loop while bit 7 on $18B is HIGH. When the output of the timer 134 goes back LOW, the adding loop is discontinued. The time e^'lapsed and, therefore, the resistance or conductance of the solution, is represented by the value of the variable in the loop. The variable value is stored in an identified location in the computer memory which has available at least ninety-six positions for storing each reading of the ninety-six electrode pairs 151 and 152 and the resistance of the numbered wells associated therewith. These values are then processed for purpose of rendering the values human-readable and displayable as discussed for the previous embodiment. As will be appreciated by those skilled in the art, the hand-held probe may be adapted to contain a multiplexing device for the selection of respective pin pairs, a processing unit to be used, for example, to generate an output of actual concentrations or other useful data, and even a miniature printer for output of the measured data on paper.

It is foreseen that the apparatus of the present invention can be adapted for use with ELISA kits that were originally intended to be read photocolormetrically and which normally include conjugate enzymes such as alkaline phosphatase or horseradish peroxidase. Such kits are available for numerous analyses, but are not directly usable for the method of the present invention. To be usable under the method of the present invention, it is necessary to have an enzyme conjugate that promotes the formation of ions in a substrate, such as urease in the manner described above.

The alkaline phosphatase and horseradish peroxidase kits may be adapted to function in conjunction with the apparatus and method of the present invention by either of two approaches.

A first approach involves the inclusion of an additional antibody reagent, such as urease-linked anti- horseradish peroxidase or urease-linked anti-alkaline phosphatase. The analysis is then initiated in the manner of an ELISA color analysis, but the additional reagent linked to urease is added in an additional step prior to testing with the apparatus of the present invention.

A second approach involves replacing the monoclonal or polyclonal detection antibodies that are conventionally linked to current enzymes, such as the horseradish peroxidase with urease-linked antibodies. The analysis would then be conducted in accordance with the methods described above. While this approach is not as easily implemented as the first approach, since a urease conjugate must be developed for each test kit analyte, it has an advantage of requiring one fewer analytical steps as compared to the first approach.

It is also foreseen that the present invention will increase the availability of ELISA technology. For example, because the apparatus of the present invention is relatively compact and very portable relative to conventional color-reading devices, the invention can be easily taken outside a laboratory to do field tests, such as on a space station (where reduced energy output is also important) or on a farm where a farmer can directly and quickly check a crop for a viral infection and take immediate corrective action. The invention also increases the range and sensitivity of the tests available and, in this manner, substances such as acquired immune deficiency syndrome (AIDS) antibodies can be detected in blood relatively very soon after infection instead of after a longer delay period with the less sensitive color-reading devices. The lower cost of the present invention as compared to color-reading devices also increases availability in third world countries and the like.

It is further foreseen that the apparatus of the present invention may be robotized, and all results computerized and automatically analyzed with a printout of such analysis, so as to provide a uniform and consistent pattern of analysis for each set of tests. Also, the conductive electrode probes may be constructed of flexible material, especially conductive plastic or rubber, to reduce the likelihood of an operator accidently pricking himself or herself with a probe used with an infectious material and to reduce mechanical problems.

As will be appreciated, the present method and apparatus are adapted to quantitatively measure immunological reactions other than those induced by enzymatic reactions but wherein, as a result of the reactions, ions are released into solution. Examples include the hybridization reaction between a natural nucleic acid and another natural or artificial nucleic acid, a hapten-antibody reaction or a biotin- avidin/streptavidin reaction. Specifically, the present invention may be used in combination with deoxyribonucleic acid (DNA) hybridization that can use DNA amplification to analyze for very small quantities of genes or DNA. For example, a conductivity reader according to the present invention is provided with a robot arm to carry out and read the conductance of the reaction solution and a temperature controlling device that is made to cycle, as required by the Taq system utilizing Taq polymerase, by a dedicated computer. A blood sample from a patient suspected of having AIDS is adequately prepared and incubated in a nitrocellulose-coated plate well. The bottom of the well is coated with nitrocellulose in the present invention since the bottom is not required to be optically transparent as with conventional color readers. The system can be used to test for HIV virus. If HIV nucleic acid is present, it will bind to the nitrocellulose. The robot arm will then add a sample of a specific primer(s) for the HIV genetic material and the reagents necessary to carry out the nucleic acid amplification. Then the computer controls the heating- cooling system. After 30-50 cycles, the viral nucleic acid sequence is amplified approximately 1,000,000 times. The robot transfers the samples into another nitrocellulose plate. The amplified nucleic acid is allowed to bind onto the nitrocellulose and then a specific HIV probe (or combination of probes) is added. These probes are tagged with biotin or a hapten (Boerhinger Mannheim) so that they can be detected by a streptavidin or anti-hapten urease conjugate. After the hybridization is completed and the conjugate has been incubated in the well, the robot arm washes the plate and adds the urea substrate. The conductivity values are then read by the apparatus of the present invention and processed by the computer. This method can be used for virtually any nucleic acid sequence. This method allows for a relatively rapid quantification of the amount of viral genetic material per volume of blood (or number of cells) and, most importantly, it does it without any human handling except the placing of the patients' samples in the plate wells.

As will be apparent to those skilled in the art, various modifications may be made in the above-described method and apparatus. It is to be understood therefore that the invention may be varied within the scope of the claims appended hereto.

Claims

I CLAIM:

1. In a method of performing an enzyme-linked immuno sorbent assay of a substance in a substrate, the improvement comprising: utilizing an enzyme conjugate with an enzyme chosen to react with said biological substance to cause the release of ions into said substrate; measuring the change in resistivity of said substrate due to the presence of said ions; generating an analog signal in response to said measurement of resistivity; converting said analog signal into a digital signal; and quantizing said digital signal and outputting same in human-readable form for indicating the quantity of said biological substance present in said substrate.

2. The method according to claim 1 wherein: said enzyme is urease and said substance is urea.

3. The method according to claim 1 wherein: said substance is initially reacted with horseradish peroxidase conjugate; and thereafter reacting said horseradish peroxidase conjugate with anti-horseradish peroxidase conjugated with said enzyme.

4. The method according to claim 3 wherein: said substance is urea and said enzyme is urease.

5. The method according to claim 1 wherein: said substrate is initially reacted with alkaline phosphatase conjugate; and thereafter reacting the alkaline phosphatase conjugate with an anti-alkaline phophatase conjugated with said enzyme.

6. The method according to claim 5 wherein: said substance is urea and said enzyme is urease.

7. The method according to claim 1 wherein: said substance is a first substance placed in a first testing well; including at least one additional substance placed in a second well; and including the step of: providing a respective set of spaced electrodes for and positioned in each substance; and sequentially applying a current to a first electrode of each set of electrodes while measuring the resistivity between each set of electrodes.

8. The method according to claim 1 including the steps of: providing a pair of electrodes located within said substance and within a well; said substance releasing ions into said well due to said enzyme in proportion to the quantity of said substance; applying a current to a first of said electrodes; generating a signal due to the conductance of the current in said substance between said electrodes; operating a timer with said signal to determine a time elapse in proportion to the conductance in said substance between said electrodes; computing a quantative amount of said substance in said well by comparing said time elapse to known quantities; and converting said quantative amount into a human- readable form.

9. In a method of performing an enzyme-linked immuno sorbent assay of an analyte in a substrate, the improvement comprising: utilizing an enzyme conjugate cooperatively chosen to react with said analyte in said substrate to produce ions within said substrate; measuring the change in resistivity of the substrate from before to after the production of said ions; and converting the change in resistivity to a human- readable form indicating the relative quantity of the analyte in the substrate.

10. Apparatus for performing an enzyme-linked immuno sorbent assay of a biological substance in a fluid substrate, comprising: means for measuring the resistivity of said substrate and producing an analog signal representative thereof; converter means for converting said analog signal into a digital signal; and processing means to process said digital signal to produce numerical data indicative of the quantity of said biological substance in said substrate.

11. The apparatus of claim 10 wherein said means for measuring the resistivity of said substrate include a plurality of pairs of electrodes for submersion in separate wells containing said substrate.

12. The apparatus of claim 11 including multiplex means for systematically selecting one of said pairs of electrodes for measurement of the resistivity of said substrate.

13. The apparatus of claim 12 further including switch means operatively connected to selected ones of said pairs of electrodes to invert the charge thereon at a predetermined rate to prevent the sustained polarization of said electrodes.

14. Apparatus for detecting the presence of a biological substance whose presence alters the resistivity/conductivity of a fluid substrate, comprising: means immersible in said substrate for measuring the resistance/conductivity of said substrate; timer means for producing a first signal at the commencement of said measurement and a second signal at the end thereof; pulse-generating means for producing a series of pulses at a steady predetermined rate; counter means enabled by said first signal and disabled by said second signal for counting said pulses produced between said first and second signals; means for converting and displaying the output of said counter means, which output is representative of the concentration of said biological substance in said substrate; and trigger means actuable to produce a first gate signal to activate said timer and a second signal to preset said counter means.

15. The apparatus of claim 14 including capacitor means connected to said timer means, said timer means generating said second signal when said capacitor means are charged to capacity.

16. The apparatus of claim 15 wherein said counter means comprise a plurality of downcounters.

17. The apparatus of claim 15 including switch means for reversing the polarity of said means for measuring at a predetermined rate.

18. The apparatus of claim 17 further including processor means for processing the output of said counting means.